%0 Journal Article %J Nature %D 2024 %T Identification of constrained sequence elements across 239 primate genomes. %A Kuderna, Lukas F K %A Ulirsch, Jacob C %A Rashid, Sabrina %A Ameen, Mohamed %A Sundaram, Laksshman %A Hickey, Glenn %A Cox, Anthony J %A Gao, Hong %A Kumar, Arvind %A Aguet, Francois %A Christmas, Matthew J %A Clawson, Hiram %A Haeussler, Maximilian %A Janiak, Mareike C %A Kuhlwilm, Martin %A Orkin, Joseph D %A Bataillon, Thomas %A Manu, Shivakumara %A Valenzuela, Alejandro %A Bergman, Juraj %A Rouselle, Marjolaine %A Silva, Felipe Ennes %A Agueda, Lidia %A Blanc, Julie %A Gut, Marta %A de Vries, Dorien %A Goodhead, Ian %A R. Alan Harris %A Raveendran, Muthuswamy %A Jensen, Axel %A Chuma, Idriss S %A Horvath, Julie E %A Hvilsom, Christina %A Juan, David %A Frandsen, Peter %A Schraiber, Joshua G %A de Melo, Fabiano R %A Bertuol, Fabrício %A Byrne, Hazel %A Sampaio, Iracilda %A Farias, Izeni %A Valsecchi, João %A Messias, Malu %A da Silva, Maria N F %A Trivedi, Mihir %A Rossi, Rogerio %A Hrbek, Tomas %A Andriaholinirina, Nicole %A Rabarivola, Clément J %A Zaramody, Alphonse %A Jolly, Clifford J %A Phillips-Conroy, Jane %A Wilkerson, Gregory %A Abee, Christian %A Simmons, Joe H %A Fernandez-Duque, Eduardo %A Kanthaswamy, Sree %A Shiferaw, Fekadu %A Wu, Dongdong %A Zhou, Long %A Shao, Yong %A Zhang, Guojie %A Keyyu, Julius D %A Knauf, Sascha %A Le, Minh D %A Lizano, Esther %A Merker, Stefan %A Navarro, Arcadi %A Nadler, Tilo %A Khor, Chiea Chuen %A Lee, Jessica %A Tan, Patrick %A Lim, Weng Khong %A Kitchener, Andrew C %A Zinner, Dietmar %A Gut, Ivo %A Melin, Amanda D %A Guschanski, Katerina %A Schierup, Mikkel Heide %A Beck, Robin M D %A Karakikes, Ioannis %A Wang, Kevin C %A Umapathy, Govindhaswamy %A Roos, Christian %A Boubli, Jean P %A Siepel, Adam %A Kundaje, Anshul %A Paten, Benedict %A Lindblad-Toh, Kerstin %A Jeffrey Rogers %A Marques Bonet, Tomas %A Farh, Kyle Kai-How %K Animals %K Conserved Sequence %K Deoxyribonuclease I %K DNA %K Evolution, Molecular %K Female %K Gene Expression Regulation %K Genome %K Humans %K Mammals %K Placenta %K Pregnancy %K Primates %K Proteins %K Regulatory Sequences, Nucleic Acid %K Reproducibility of Results %K Transcription Factors %X

Noncoding DNA is central to our understanding of human gene regulation and complex diseases, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA, the relatively short timescales separating primate species, and the previously limited availability of whole-genome sequences. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

%B Nature %V 625 %P 735-742 %8 2024 Jan %G eng %N 7996 %1 https://www.ncbi.nlm.nih.gov/pubmed/38030727?dopt=Abstract %R 10.1038/s41586-023-06798-8 %0 Journal Article %J JAMA Cardiol %D 2023 %T Association of Rare Protein-Truncating DNA Variants in APOB or PCSK9 With Low-density Lipoprotein Cholesterol Level and Risk of Coronary Heart Disease. %A Dron, Jacqueline S %A Patel, Aniruddh P %A Zhang, Yiyi %A Jurgens, Sean J %A Maamari, Dimitri J %A Wang, Minxian %A Eric Boerwinkle %A Morrison, Alanna C %A de Vries, Paul S %A Fornage, Myriam %A Hou, Lifang %A Lloyd-Jones, Donald M %A Psaty, Bruce M %A Tracy, Russell P %A Bis, Joshua C %A Vasan, Ramachandran S %A Levy, Daniel %A Heard-Costa, Nancy %A Rich, Stephen S %A Guo, Xiuqing %A Taylor, Kent D %A Richard A Gibbs %A Rotter, Jerome I %A Willer, Cristen J %A Oelsner, Elizabeth C %A Moran, Andrew E %A Peloso, Gina M %A Natarajan, Pradeep %A Khera, Amit V %K Adult %K Aged %K Apolipoproteins B %K Cholesterol, LDL %K Coronary Disease %K DNA %K Female %K Humans %K Male %K Middle Aged %K Proprotein Convertase 9 %K Prospective Studies %X

IMPORTANCE: Protein-truncating variants (PTVs) in apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with significantly lower low-density lipoprotein (LDL) cholesterol concentrations. The association of these PTVs with coronary heart disease (CHD) warrants further characterization in large, multiracial prospective cohort studies.

OBJECTIVE: To evaluate the association of PTVs in APOB and PCSK9 with LDL cholesterol concentrations and CHD risk.

DESIGN, SETTING, AND PARTICIPANTS: This studied included participants from 5 National Heart, Lung, and Blood Institute (NHLBI) studies and the UK Biobank. NHLBI study participants aged 5 to 84 years were recruited between 1971 and 2002 across the US and underwent whole-genome sequencing. UK Biobank participants aged 40 to 69 years were recruited between 2006 and 2010 in the UK and underwent whole-exome sequencing. Data were analyzed from June 2021 to October 2022.

EXPOSURES: PTVs in APOB and PCSK9.

MAIN OUTCOMES AND MEASURES: Estimated untreated LDL cholesterol levels and CHD.

RESULTS: Among 19 073 NHLBI participants (10 598 [55.6%] female; mean [SD] age, 52 [17] years), 139 (0.7%) carried an APOB or PCSK9 PTV, which was associated with 49 mg/dL (95% CI, 43-56) lower estimated untreated LDL cholesterol level. Over a median (IQR) follow-up of 21.5 (13.9-29.4) years, incident CHD was observed in 12 of 139 carriers (8.6%) vs 3029 of 18 934 noncarriers (16.0%), corresponding to an adjusted hazard ratio of 0.51 (95% CI, 0.28-0.89; P = .02). Among 190 464 UK Biobank participants (104 831 [55.0%] female; mean [SD] age, 57 [8] years), 662 (0.4%) carried a PTV, which was associated with 45 mg/dL (95% CI, 42-47) lower estimated untreated LDL cholesterol level. Estimated CHD risk by age 75 years was 3.7% (95% CI, 2.0-5.3) in carriers vs 7.0% (95% CI, 6.9-7.2) in noncarriers, corresponding to an adjusted hazard ratio of 0.51 (95% CI, 0.32-0.81; P = .004).

CONCLUSIONS AND RELEVANCE: Among 209 537 individuals in this study, 0.4% carried an APOB or PCSK9 PTV that was associated with less exposure to LDL cholesterol and a 49% lower risk of CHD.

%B JAMA Cardiol %V 8 %P 258-267 %8 2023 Mar 01 %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/36723951?dopt=Abstract %R 10.1001/jamacardio.2022.5271 %0 Journal Article %J J Agric Food Chem %D 2022 %T Isolation and Barcoding of Trace Pollen-free DNA for Authentication of Honey. %A Chavan, Dimple %A Adolacion, Jay R T %A Crum, Mary %A Nandy, Suman %A Lee, Kyung Hyun %A Vu, Binh %A Kourentzi, Katerina %A Aniko Sabo %A Willson, Richard C %K DNA %K DNA Barcoding, Taxonomic %K Honey %K Pollen %X

Adulteration and mislabeling of honey to mask its true origin have become a global concern. Pollen microscopy, the current gold standard for identifying honey's geographical and plant origins, is laborious, requires extensive training, and fails to identify filtered honey and honey spiked with pollen from a more favorable plant to disguise its origins. We successfully isolated pollen-free DNA from filtered honey using three types of adsorbents: (i) anti-dsDNA antibodies coupled to magnetic microspheres; (ii) anion-exchange adsorbent; and (iii) ceramic hydroxyapatite. The internal transcribed spacer 2 region of the captured pollen-free DNA was polymerase chain reaction-amplified and subjected to next-generation sequencing. Using an in-house bioinformatics pipeline, initial experiments showed that anion exchange had the greatest capacity to capture trace pollen-free DNA, and it was successfully applied to isolate DNA from five honey samples. Enrichment of trace pollen-free DNA from filtered honey samples opens a new approach for identifying the true origins of honey.

%B J Agric Food Chem %V 70 %P 14084-14095 %8 2022 Nov 02 %G eng %N 43 %1 https://www.ncbi.nlm.nih.gov/pubmed/36279293?dopt=Abstract %R 10.1021/acs.jafc.2c04309 %0 Journal Article %J Genome Med %D 2022 %T The multiple de novo copy number variant (MdnCNV) phenomenon presents with peri-zygotic DNA mutational signatures and multilocus pathogenic variation. %A Du, Haowei %A Jolly, Angad %A Grochowski, Christopher M %A Bo Yuan %A Dawood, Moez %A Jhangiani, Shalini N %A Li, He %A Donna M Muzny %A Fatih, Jawid M %A Coban-Akdemir, Zeynep %A Carlin, Mary Esther %A Scheuerle, Angela E %A Witzl, Karin %A Posey, Jennifer E %A Pendleton, Matthew %A Harrington, Eoghan %A Juul, Sissel %A Hastings, P J %A Bi, Weimin %A Richard A Gibbs %A Fritz J Sedlazeck %A James R Lupski %A Carvalho, Claudia M B %A Liu, Pengfei %K Comparative Genomic Hybridization %K DNA %K DNA Copy Number Variations %K DNA-Binding Proteins %K Genomic Instability %K Humans %K Mutation %K Nucleotides %K Transcription Factors %X

BACKGROUND: The multiple de novo copy number variant (MdnCNV) phenotype is described by having four or more constitutional de novo CNVs (dnCNVs) arising independently throughout the human genome within one generation. It is a rare peri-zygotic mutational event, previously reported to be seen once in every 12,000 individuals referred for genome-wide chromosomal microarray analysis due to congenital abnormalities. These rare families provide a unique opportunity to understand the genetic factors of peri-zygotic genome instability and the impact of dnCNV on human diseases.

METHODS: Chromosomal microarray analysis (CMA), array-based comparative genomic hybridization, short- and long-read genome sequencing (GS) were performed on the newly identified MdnCNV family to identify de novo mutations including dnCNVs, de novo single-nucleotide variants (dnSNVs), and indels. Short-read GS was performed on four previously published MdnCNV families for dnSNV analysis. Trio-based rare variant analysis was performed on the newly identified individual and four previously published MdnCNV families to identify potential genetic etiologies contributing to the peri-zygotic genomic instability. Lin semantic similarity scores informed quantitative human phenotype ontology analysis on three MdnCNV families to identify gene(s) driving or contributing to the clinical phenotype.

RESULTS: In the newly identified MdnCNV case, we revealed eight de novo tandem duplications, each ~ 1 Mb, with microhomology at 6/8 breakpoint junctions. Enrichment of de novo single-nucleotide variants (SNV; 6/79) and de novo indels (1/12) was found within 4 Mb of the dnCNV genomic regions. An elevated post-zygotic SNV mutation rate was observed in MdnCNV families. Maternal rare variant analyses identified three genes in distinct families that may contribute to the MdnCNV phenomenon. Phenotype analysis suggests that gene(s) within dnCNV regions contribute to the observed proband phenotype in 3/3 cases. CNVs in two cases, a contiguous gene duplication encompassing PMP22 and RAI1 and another duplication affecting NSD1 and SMARCC2, contribute to the clinically observed phenotypic manifestations.

CONCLUSIONS: Characteristic features of dnCNVs reported here are consistent with a microhomology-mediated break-induced replication (MMBIR)-driven mechanism during the peri-zygotic period. Maternal genetic variants in DNA repair genes potentially contribute to peri-zygotic genomic instability. Variable phenotypic features were observed across a cohort of three MdnCNV probands, and computational quantitative phenotyping revealed that two out of three had evidence for the contribution of more than one genetic locus to the proband's phenotype supporting the hypothesis of de novo multilocus pathogenic variation (MPV) in those families.

%B Genome Med %V 14 %P 122 %8 2022 Oct 27 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/36303224?dopt=Abstract %R 10.1186/s13073-022-01123-w %0 Journal Article %J Nucleic Acids Res %D 2021 %T High resolution copy number inference in cancer using short-molecule nanopore sequencing. %A Baslan, Timour %A Kovaka, Sam %A Fritz J Sedlazeck %A Zhang, Yanming %A Wappel, Robert %A Tian, Sha %A Lowe, Scott W %A Goodwin, Sara %A Schatz, Michael C %K Cell Line, Tumor %K DNA %K DNA Copy Number Variations %K Humans %K Medical Oncology %K Nanopore Sequencing %K Neoplasms %X

Genome copy number is an important source of genetic variation in health and disease. In cancer, Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms, limiting CNA inference accuracy. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that short-molecule nanopore sequencing reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for short-molecule nanopore sequencing with applications in research and medicine, which includes but is not limited to, CNAs.

%B Nucleic Acids Res %V 49 %P e124 %8 2021 Dec 02 %G eng %N 21 %1 https://www.ncbi.nlm.nih.gov/pubmed/34551429?dopt=Abstract %R 10.1093/nar/gkab812 %0 Journal Article %J J Virol %D 2021 %T Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA. %A Smith, Lisa M %A Ladner, Jason T %A Hodara, Vida L %A Parodi, Laura M %A R. Alan Harris %A Callery, Jessica E %A Lai, Zhao %A Zou, Yi %A Raveedran, Muthuswamy %A Jeffrey Rogers %A Giavedoni, Luis D %K Animals %K CRISPR-Cas Systems %K Deoxyribonuclease I %K DNA %K Endonucleases %K Gene Editing %K HEK293 Cells %K Humans %K Macaca mulatta %K Mutagenesis %K Plasmids %K Proviruses %K RNA, Guide, Kinetoplastida %K Simian Immunodeficiency Virus %X

Human and simian immunodeficiency virus (HIV and SIV) infections establish lifelong reservoirs of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactivate integrated provirus DNA and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells cotransfected with SIV provirus and plasmids coding for RGNus identified SIV long terminal repeat (LTR), -activation response (TAR) element, and ribosome slip site (RSS) regions as the most effective at virus suppression; RGNi targeting these regions inhibited virus production significantly. Multiplex plasmids that coexpressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIV, and whole-genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations, making these tools a desirable prospect for moving into animal studies. Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole-genome sequencing demonstrated effective SIV sequence mutation and inactivation and the absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.

%B J Virol %V 95 %P e0088221 %8 2021 Nov 09 %G eng %N 23 %1 https://www.ncbi.nlm.nih.gov/pubmed/34549979?dopt=Abstract %R 10.1128/JVI.00882-21 %0 Journal Article %J Nat Biotechnol %D 2021 %T Performance assessment of DNA sequencing platforms in the ABRF Next-Generation Sequencing Study. %A Foox, Jonathan %A Tighe, Scott W %A Nicolet, Charles M %A Zook, Justin M %A Byrska-Bishop, Marta %A Clarke, Wayne E %A Khayat, Michael M %A Mahmoud, Medhat %A Laaguiby, Phoebe K %A Herbert, Zachary T %A Warner, Derek %A Grills, George S %A Jen, Jin %A Levy, Shawn %A Xiang, Jenny %A Alonso, Alicia %A Zhao, Xia %A Zhang, Wenwei %A Teng, Fei %A Zhao, Yonggang %A Lu, Haorong %A Schroth, Gary P %A Narzisi, Giuseppe %A Farmerie, William %A Fritz J Sedlazeck %A Baldwin, Don A %A Mason, Christopher E %K Base Pair Mismatch %K Benchmarking %K DNA %K DNA, Bacterial %K Genome, Bacterial %K Genome, Human %K High-Throughput Nucleotide Sequencing %K Humans %K Sequence Analysis, DNA %X

Assessing the reproducibility, accuracy and utility of massively parallel DNA sequencing platforms remains an ongoing challenge. Here the Association of Biomolecular Resource Facilities (ABRF) Next-Generation Sequencing Study benchmarks the performance of a set of sequencing instruments (HiSeq/NovaSeq/paired-end 2 × 250-bp chemistry, Ion S5/Proton, PacBio circular consensus sequencing (CCS), Oxford Nanopore Technologies PromethION/MinION, BGISEQ-500/MGISEQ-2000 and GS111) on human and bacterial reference DNA samples. Among short-read instruments, HiSeq 4000 and X10 provided the most consistent, highest genome coverage, while BGI/MGISEQ provided the lowest sequencing error rates. The long-read instrument PacBio CCS had the highest reference-based mapping rate and lowest non-mapping rate. The two long-read platforms PacBio CCS and PromethION/MinION showed the best sequence mapping in repeat-rich areas and across homopolymers. NovaSeq 6000 using 2 × 250-bp read chemistry was the most robust instrument for capturing known insertion/deletion events. This study serves as a benchmark for current genomics technologies, as well as a resource to inform experimental design and next-generation sequencing variant calling.

%B Nat Biotechnol %V 39 %P 1129-1140 %8 2021 Sep %G eng %N 9 %1 https://www.ncbi.nlm.nih.gov/pubmed/34504351?dopt=Abstract %R 10.1038/s41587-021-01049-5 %0 Journal Article %J Circulation %D 2020 %T Critical Role of Cytosolic DNA and Its Sensing Adaptor STING in Aortic Degeneration, Dissection, and Rupture. %A Luo, Wei %A Wang, Yidan %A Zhang, Lin %A Ren, Pingping %A Zhang, Chen %A Li, Yanming %A Azares, Alon R %A Zhang, Michelle %A Guo, Jiao %A Ghaghada, Ketan B %A Starosolski, Zbigniew A %A Rajapakshe, Kimal %A Coarfa, Cristian %A Yumei Li %A Rui Chen %A Fujiwara, Keigi %A Abe, Jun-Ichi %A Coselli, Joseph S %A Milewicz, Dianna M %A LeMaire, Scott A %A Shen, Ying H %K Animals %K Aortic Dissection %K Aortic Rupture %K Cytosol %K DNA %K Female %K Male %K Membrane Proteins %K Mice %K Mice, Knockout %K Signal Transduction %X

BACKGROUND: Sporadic aortic aneurysm and dissection (AAD), caused by progressive aortic smooth muscle cell (SMC) loss and extracellular matrix degradation, is a highly lethal condition. Identifying mechanisms that drive aortic degeneration is a crucial step in developing an effective pharmacologic treatment to prevent disease progression. Recent evidence has indicated that cytosolic DNA and abnormal activation of the cytosolic DNA sensing adaptor STING (stimulator of interferon genes) play a critical role in vascular inflammation and destruction. Here, we examined the involvement of this mechanism in aortic degeneration and sporadic AAD formation.

METHODS: The presence of cytosolic DNA in aortic cells and activation of the STING pathway were examined in aortic tissues from patients with sporadic ascending thoracic AAD. The role of STING in AAD development was evaluated in -deficient () mice in a sporadic AAD model induced by challenging mice with a combination of a high-fat diet and angiotensin II. We also examined the direct effects of STING on SMC death and macrophage activation in vitro.

RESULTS: In human sporadic AAD tissues, we observed the presence of cytosolic DNA in SMCs and macrophages and significant activation of the STING pathway. In the sporadic AAD model, mice showed significant reductions in challenge-induced aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions. Single-cell transcriptome analysis revealed that aortic challenge in wild-type mice induced the DNA damage response, the inflammatory response, dedifferentiation and cell death in SMCs, and matrix metalloproteinase expression in macrophages. These changes were attenuated in challenged mice. Mechanistically, nuclear and mitochondrial DNA damage in SMCs and the subsequent leak of DNA to the cytosol activated STING signaling, which induced cell death through apoptosis and necroptosis. In addition, DNA from damaged SMCs was engulfed by macrophages in which it activated STING and its target interferon regulatory factor 3, which directly induced matrix metalloproteinase-9 expression. We also found that pharmacologically inhibiting STING activation partially prevented AAD development.

CONCLUSIONS: Our findings indicate that the presence of cytosolic DNA and subsequent activation of cytosolic DNA sensing adaptor STING signaling represent a key mechanism in aortic degeneration and that targeting STING may prevent sporadic AAD development.

%B Circulation %V 141 %P 42-66 %8 2020 Jan 07 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/31887080?dopt=Abstract %R 10.1161/CIRCULATIONAHA.119.041460 %0 Journal Article %J Nat Methods %D 2020 %T Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing. %A Lee, Isac %A Razaghi, Roham %A Gilpatrick, Timothy %A Molnar, Michael %A Gershman, Ariel %A Sadowski, Norah %A Fritz J Sedlazeck %A Hansen, Kasper D %A Simpson, Jared T %A Timp, Winston %K Breast Neoplasms %K Cell Line, Tumor %K Chromatin %K CpG Islands %K DNA %K DNA Methylation %K Epigenome %K Female %K Genome, Human %K Humans %K MCF-7 Cells %K Methyltransferases %K Nanopore Sequencing %K Promoter Regions, Genetic %K Sequence Analysis, DNA %X

Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of nucleosome occupancy and methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7 and MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding, and determination of the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate a fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and noncancerous cells.

%B Nat Methods %V 17 %P 1191-1199 %8 2020 Dec %G eng %N 12 %1 https://www.ncbi.nlm.nih.gov/pubmed/33230324?dopt=Abstract %R 10.1038/s41592-020-01000-7 %0 Journal Article %J Nat Commun %D 2019 %T Subtle changes in chromatin loop contact propensity are associated with differential gene regulation and expression. %A Greenwald, William W %A Li, He %A Benaglio, Paola %A Jakubosky, David %A Matsui, Hiroko %A Schmitt, Anthony %A Selvaraj, Siddarth %A D'Antonio, Matteo %A D'Antonio-Chronowska, Agnieszka %A Smith, Erin N %A Frazer, Kelly A %K Adolescent %K Adult %K Aged %K Cell Line %K Chromatin %K DNA %K Female %K Gene Expression Regulation %K Histones %K Humans %K Induced Pluripotent Stem Cells %K Male %K Middle Aged %K Myocytes, Cardiac %K Nucleic Acid Conformation %K Polymorphism, Single Nucleotide %K Quantitative Trait Loci %K Whole Genome Sequencing %K Young Adult %X

While genetic variation at chromatin loops is relevant for human disease, the relationships between contact propensity (the probability that loci at loops physically interact), genetics, and gene regulation are unclear. We quantitatively interrogate these relationships by comparing Hi-C and molecular phenotype data across cell types and haplotypes. While chromatin loops consistently form across different cell types, they have subtle quantitative differences in contact frequency that are associated with larger changes in gene expression and H3K27ac. For the vast majority of loci with quantitative differences in contact frequency across haplotypes, the changes in magnitude are smaller than those across cell types; however, the proportional relationships between contact propensity, gene expression, and H3K27ac are consistent. These findings suggest that subtle changes in contact propensity have a biologically meaningful role in gene regulation and could be a mechanism by which regulatory genetic variants in loop anchors mediate effects on expression.

%B Nat Commun %V 10 %P 1054 %8 2019 Mar 05 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/30837461?dopt=Abstract %R 10.1038/s41467-019-08940-5 %0 Journal Article %J Biopreserv Biobank %D 2018 %T National Cancer Institute Biospecimen Evidence-Based Practices: Harmonizing Procedures for Nucleic Acid Extraction from Formalin-Fixed, Paraffin-Embedded Tissue. %A Greytak, Sarah R %A Engel, Kelly B %A Zmuda, Erik %A Casas-Silva, Esmeralda %A Guan, Ping %A Hoadley, Katherine A %A Mungall, Andrew J %A Wheeler, David A %A Doddapaneni, Harsha V %A Moore, Helen M %K DNA %K Evidence-Based Practice %K Gene Library %K Genomics %K Humans %K National Cancer Institute (U.S.) %K Nucleic Acids %K Paraffin Embedding %K RNA %K United States %B Biopreserv Biobank %V 16 %P 247-250 %8 2018 Aug %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/29920119?dopt=Abstract %R 10.1089/bio.2018.0046 %0 Journal Article %J BMC Med Genet %D 2017 %T Identification of established arrhythmogenic right ventricular cardiomyopathy mutation in a patient with the contrasting phenotype of hypertrophic cardiomyopathy. %A Bainbridge, Matthew Neil %A Li, Lili %A Tan, Yanli %A Cheong, Benjamin Y %A Marian, Ali J %K Cardiomyopathy, Hypertrophic %K DNA %K DNA Mutational Analysis %K Echocardiography %K Female %K Heart %K Heart Ventricles %K Humans %K Magnetic Resonance Imaging %K Middle Aged %K Pedigree %K Phenotype %K Plakophilins %X

BACKGROUND: Advances in the nucleic acid sequencing technologies have ushered in the era of genetic-based "precision medicine". Applications of the genetic discoveries to practice of medicine, however, are hindered by phenotypic variability of the genetic variants. The report illustrates extreme pleiotropic phenotypes associated with an established causal mutation for hereditary cardiomyopathy.

CASE PRESENTATION: We report a 61-year old white female who presented with syncope and echocardiographic and cardiac magnetic resonance (CMR) imaging findings consistent with the diagnosis of hypertrophic cardiomyopathy (HCM). The electrocardiogram, however, showed a QRS pattern resembling an Epsilon wave, a feature of arrhythmogenic right ventricular cardiomyopathy (ARVC). Whole exome sequencing (mean depth of coverage of exons 178X) analysis did not identify a pathogenic variant in the known HCM genes but identified an established causal mutation for ARVC. The mutation involves a canonical splice accepter site (c.2146-1G > C) in the PKP2 gene, which encodes plakophillin 2. Sanger sequencing confirmed the mutation. PKP2 is the most common causal gene for ARVC but has not been implicated in HCM. Findings on echocardiography and CMR during the course of 4-year follow up showed septal hypertrophy and a hyperdynamic left ventricle, consistent with the diagnosis of HCM. However, neither baseline nor follow up echocardiography and CMR studies showed evidence of ARVC. The right ventricle was normal in size, thickness, and function and there was no evidence of fibro-fatty infiltration in the myocardium.

CONCLUSIONS: The patient carries an established pathogenic mutation for ARVC and a subtle finding of ARVC but exhibits the classic phenotype of HCM, a contrasting phenotype to ARVC. The case illustrates the need for detailed phenotypic characterization for patients with hereditary cardiomyopathies as well as the challenges physicians face in applying the genetic discoveries in practicing genetic-based "precision medicine".

%B BMC Med Genet %V 18 %P 24 %8 2017 Mar 03 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/28253841?dopt=Abstract %R 10.1186/s12881-017-0385-8 %0 Journal Article %J Invest Ophthalmol Vis Sci %D 2017 %T IFT81 as a Candidate Gene for Nonsyndromic Retinal Degeneration. %A Dharmat, Rachayata %A Liu, Wei %A Ge, Zhongqi %A Sun, Zixi %A Yang, Lizhu %A Li, Yumei %A Wang, Keqing %A Thomas, Kandace %A Sui, Ruifang %A Chen, Rui %K Animals %K Cells, Cultured %K Codon, Nonsense %K Disease Models, Animal %K DNA %K DNA Mutational Analysis %K Female %K Humans %K Male %K Muscle Proteins %K Mutation %K Phenotype %K Retina %K Retinal Degeneration %K Zebrafish %X

PURPOSE: IFT81, a core component of the IFT-B complex, involved in the bidirectional transport of ciliary proteins, has been recently implicated in syndromic ciliopathies. However, none of the IFT-B core complex proteins have been associated with nonsyndromic retinal dystrophies. Given the importance of ciliary transport in photoreceptor function and structural maintenance, we sought to investigate the impact of IFT (intraflagellar transport) mutations in nonsyndromic retinopathies.

METHODS: Whole exome sequencing was performed on 50 cone-rod dystrophy (CRD) patients that were previously screened for mutations in known retinal disease genes. The impact of candidate mutation was studied using in vitro cell system and in vivo zebrafish assay to determine the pathogenicity of the variant.

RESULTS: Compound heterozygous mutations in IFT81, including one nonsense (c.1213C>T, p.R405*) and one missense variant (c.1841T>C, p.L614P), were identified in a nonsyndromic CRD proband. Extensive functional analyses of the missense variant in cell culture and zebrafish strongly suggests its pathogenic nature. Loss of IFT81 impairs ciliogenesis and, interestingly, the missense variant displayed significantly reduced rescue of ciliogenesis in the IFT81 knockdown in vitro system. Consistently, dramatic reduction of rescue efficiency of the ift81 mutant zebrafish embryo by mRNA with the missense variant was observed, further supporting its pathogenicity.

CONCLUSIONS: Consistent with the function of the IFT-B complex in the maintenance of photoreceptor cilium, we report a case of mutations in a core IFT-B protein, IFT81. This represents the first report of mutations in IFT81 as a candidate gene for nonsyndromic retinal dystrophy, hence expanding the phenotype spectrum of IFT-B components.

%B Invest Ophthalmol Vis Sci %V 58 %P 2483-2490 %8 2017 May 01 %G eng %N 5 %1 https://www.ncbi.nlm.nih.gov/pubmed/28460050?dopt=Abstract %R 10.1167/iovs.16-19133 %0 Journal Article %J Invest Ophthalmol Vis Sci %D 2014 %T A homozygous missense mutation in NEUROD1 is associated with nonsyndromic autosomal recessive retinitis pigmentosa. %A Wang, Feng %A Li, Huajin %A Xu, Mingchu %A Li, Hui %A Zhao, Li %A Yang, Lizhu %A Zaneveld, Jacques E %A Wang, Keqing %A Li, Yumei %A Sui, Ruifang %A Chen, Rui %K Adult %K Basic Helix-Loop-Helix Transcription Factors %K DNA %K DNA Mutational Analysis %K Female %K Genes, Recessive %K Homozygote %K Humans %K Male %K Mutation, Missense %K Pedigree %K Phenotype %K Retinitis Pigmentosa %X

PURPOSE: Mutations in the same gene can lead to different clinical phenotypes. In this study, we aim to identify novel genotype-phenotype correlations and novel disease genes by analyzing an unsolved autosomal recessive retinitis pigmentosa (ARRP) Han Chinese family.

METHODS: Whole exome sequencing was performed for one proband from the consanguineous ARRP family. Stringent variants filtering and prioritizations were applied to identify the causative mutation.

RESULTS: A homozygous missense variant, c.724G>A; p.V242I, in NEUROD1 was identified as the most likely cause of disease. This allele perfectly segregates in the family and affects an amino acid, which is highly conserved among mammals. A previous study showed that a homozygous null allele in NEUROD1 causes severe syndromic disease with neonatal diabetes, systematic neurological abnormalities, and early-onset retinal dystrophy. Consistent with these results, our patients who are homozygous for a less severe missense allele presented only late-onset retinal degeneration without any syndromic symptoms.

CONCLUSIONS: We identified a potential novel genotype-phenotype correlation between NEUROD1 and nonsyndromic ARRP. Our study supports the idea that NEUROD1 is important for maintenance of the retina function and partial loss-of-function mutation in NEUROD1 is likely a rare cause of nonsyndromic ARRP.

%B Invest Ophthalmol Vis Sci %V 56 %P 150-5 %8 2014 Dec 04 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/25477324?dopt=Abstract %R 10.1167/iovs.14-15382 %0 Journal Article %J Invest Ophthalmol Vis Sci %D 2014 %T A missense mutation in HK1 leads to autosomal dominant retinitis pigmentosa. %A Wang, Feng %A Wang, Yandong %A Zhang, Bin %A Zhao, Li %A Lyubasyuk, Vera %A Wang, Keqing %A Xu, Mingchu %A Li, Yumei %A Wu, Frances %A Wen, Cindy %A Bernstein, Paul S %A Lin, Danni %A Zhu, Susanna %A Wang, Hui %A Zhang, Kang %A Chen, Rui %K Adolescent %K Child %K Child, Preschool %K DNA %K DNA Mutational Analysis %K Female %K Genes, Dominant %K Genetic Linkage %K Genetic Predisposition to Disease %K Heterozygote %K Hexokinase %K Humans %K Male %K Mutation, Missense %K Pedigree %K Phenotype %K Retinitis Pigmentosa %K Young Adult %X

PURPOSE: Retinitis pigmentosa (RP) is a genetically heterogeneous disease with over 60 causative genes known to date. Nevertheless, approximately 40% of RP cases remain genetically unsolved, suggesting that many novel disease-causing genes are yet to be identified. In this study, we aimed to identify the causative mutation for a large autosomal dominant RP (adRP) family with negative results from known retinal disease gene screening.

METHODS: Linkage analysis followed by whole-exome sequencing was performed. Stringent variant filtering and prioritization was carried out to identify the causative mutation.

RESULTS: Linkage analysis identified a minimal disease region of 8 Mb on chromosome 10 with a peak parametric logarithm (base 10) of odds (LOD) score of 3.500. Further whole-exome sequencing identified a heterozygous missense mutation (NM_000188.2:c.2539G>A, p.E847K) in hexokinase 1 (HK1) that segregated with the disease phenotype in the family. Biochemical assays showed that the E847K mutation does not affect hexokinase enzymatic activity or the protein stability, suggesting that the mutation may impact other uncharacterized function or result in a gain of function of HK1.

CONCLUSIONS: Here, we identified HK1 as a novel causative gene for adRP. This is the first report that associates the glucose metabolic pathway with human retinal degenerative disease, suggesting a potential new disease mechanism.

%B Invest Ophthalmol Vis Sci %V 55 %P 7159-64 %8 2014 Oct 14 %G eng %N 11 %1 https://www.ncbi.nlm.nih.gov/pubmed/25316723?dopt=Abstract %R 10.1167/iovs.14-15520 %0 Journal Article %J Genome Res %D 2013 %T DANPOS: dynamic analysis of nucleosome position and occupancy by sequencing. %A Chen, Kaifu %A Xi, Yuanxin %A Pan, Xuewen %A Li, Zhaoyu %A Kaestner, Klaus %A Tyler, Jessica %A Dent, Sharon %A He, Xiangwei %A Li, Wei %K Algorithms %K Animals %K Computational Biology %K Computer Simulation %K Databases, Genetic %K DNA %K High-Throughput Nucleotide Sequencing %K Humans %K Mice %K Nucleosomes %K Promoter Regions, Genetic %K Protein Binding %K ROC Curve %X

Recent developments in next-generation sequencing have enabled whole-genome profiling of nucleosome organizations. Although several algorithms for inferring nucleosome position from a single experimental condition have been available, it remains a challenge to accurately define dynamic nucleosomes associated with environmental changes. Here, we report a comprehensive bioinformatics pipeline, DANPOS, explicitly designed for dynamic nucleosome analysis at single-nucleotide resolution. Using both simulated and real nucleosome data, we demonstrated that bias correction in preliminary data processing and optimal statistical testing significantly enhances the functional interpretation of dynamic nucleosomes. The single-nucleotide resolution analysis of DANPOS allows us to detect all three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework. Pathway analysis indicates that each category is involved in distinct biological functions. We also analyzed the influence of sequencing depth and suggest that even 200-fold coverage is probably not enough to identify all the dynamic nucleosomes. Finally, based on nucleosome data from the human hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ESCs), we demonstrated that DANPOS is also robust in defining functional dynamic nucleosomes, not only in promoters, but also in distal regulatory regions in the mammalian genome.

%B Genome Res %V 23 %P 341-51 %8 2013 Feb %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/23193179?dopt=Abstract %R 10.1101/gr.142067.112 %0 Journal Article %J J Med Primatol %D 2012 %T Androgen receptor CAG repeat polymorphism in males of six non-human primate species. %A Mubiru, James N %A Cavazos, Nicole %A Hemmat, Peggah %A Garcia-Forey, Magdalena %A Shade, Robert E %A Rogers, Jeffrey %K Animals %K DNA %K Male %K Pan troglodytes %K Polymerase Chain Reaction %K Polymorphism, Genetic %K Primates %K Receptors, Androgen %K Trinucleotide Repeats %X

BACKGROUND: Androgen receptor [CAG](n) microsatellite has been linked to human diseases.

METHODS: Six non-human primates were genotyped for the [CAG](n) microsatellite.

RESULTS: Marmosets and macaques are monomorphic, while mangabeys, baboons, and chimpanzees are polymorphic.

CONCLUSIONS: Non-human primates that are polymorphic for the microsatellite are candidate animal models for CAG-related diseases.

%B J Med Primatol %V 41 %P 67-70 %8 2012 Feb %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/21978295?dopt=Abstract %R 10.1111/j.1600-0684.2011.00517.x %0 Journal Article %J Hum Mutat %D 2012 %T The KAT6B-related disorders genitopatellar syndrome and Ohdo/SBBYS syndrome have distinct clinical features reflecting distinct molecular mechanisms. %A Campeau, Philippe M %A Lu, James T %A Dawson, Brian C %A Fokkema, Ivo F A C %A Robertson, Stephen P %A Gibbs, Richard A %A Lee, Brendan H %K Abnormalities, Multiple %K Base Sequence %K Blepharophimosis %K Blepharoptosis %K Craniofacial Abnormalities %K Databases, Nucleic Acid %K DNA %K Female %K Genetic Association Studies %K Haploinsufficiency %K Heart Defects, Congenital %K Histone Acetyltransferases %K Humans %K Intellectual Disability %K Kidney %K Male %K Molecular Sequence Data %K Mutation %K Patella %K Psychomotor Disorders %K Scrotum %K Sequence Deletion %K Urogenital Abnormalities %X

Genitopatellar syndrome (GPS) and Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) have both recently been shown to be caused by distinct mutations in the histone acetyltransferase KAT6B (a.k.a. MYST4/MORF). All variants are de novo dominant mutations that lead to protein truncation. Mutations leading to GPS occur in the proximal portion of the last exon and lead to the expression of a protein without a C-terminal domain. Mutations leading to SBBYSS occur either throughout the gene, leading to nonsense-mediated decay, or more distally in the last exon. Features present only in GPS are contractures, anomalies of the spine, ribs and pelvis, renal cysts, hydronephrosis, and agenesis of the corpus callosum. Features present only in SBBYSS include long thumbs and long great toes and lacrimal duct abnormalities. Several features occur in both, such as intellectual disability, congenital heart defects, and genital and patellar anomalies. We propose that haploinsufficiency or loss of a function mediated by the C-terminal domain causes the common features, whereas gain-of-function activities would explain the features unique to GPS. Further molecular studies and the compilation of mutations in a database for genotype-phenotype correlations (www.LOVD.nl/KAT6B) might help tease out answers to these questions and understand the developmental programs dysregulated by the different truncations.

%B Hum Mutat %V 33 %P 1520-5 %8 2012 Nov %G eng %N 11 %1 https://www.ncbi.nlm.nih.gov/pubmed/22715153?dopt=Abstract %R 10.1002/humu.22141 %0 Journal Article %J Atherosclerosis %D 2011 %T Global DNA methylation and risk of subclinical atherosclerosis in young adults: the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. %A Bressler, Jan %A Shimmin, Lawrence C %A Boerwinkle, Eric %A Hixson, James E %K Adolescent %K Adult %K Age Factors %K Aorta, Abdominal %K Aorta, Thoracic %K Aortic Diseases %K Asymptomatic Diseases %K Atherosclerosis %K Autopsy %K Black or African American %K Case-Control Studies %K Coronary Artery Disease %K Coronary Vessels %K DNA %K DNA Methylation %K Female %K Humans %K Liver %K Logistic Models %K Male %K Matched-Pair Analysis %K Odds Ratio %K Plaque, Atherosclerotic %K Risk Assessment %K Risk Factors %K Texas %K White People %K Young Adult %X

OBJECTIVE: The association between hepatic global DNA methylation measured using pyrosequencing technology and the risk of subclinical atherosclerosis was examined in the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. PDAY is a bi-racial investigation of the natural history of atherosclerosis and its risk factors involving 3013 individuals aged 15-34 years who underwent autopsy after dying of unrelated causes in 1987-1994.

METHODS: Raised atherosclerotic lesions were defined as the sum of the percentages of intimal surface area detected in the right coronary artery and left half of the abdominal and thoracic aorta harboring fibrous plaques, complicated lesions, and calcified lesions during a postmortem pathological examination. To conduct the case-control study, 300 cases selected with the highest raised lesion scores were paired with 300 controls without raised lesions after matching for age, race, and gender.

RESULTS: Global DNA methylation was not associated with disease risk in the study population considered as a whole using conditional logistic regression models to analyze matched pairs. Since the estimation of the risk of atherosclerosis associated with inter-individual variation in DNA methylation was similar if unconditional logistic regression was used, subgroup analyses were carried out after adjusting for matching variables. A modest association with methylation levels below the median value was found in white but not in African-American study participants (odds ratio = 1.59, 95% confidence interval = 1.02-2.49, p = 0.04).

CONCLUSIONS: Hepatic global DNA methylation does not appear to be a definitive determinant of atherosclerosis burden in a postmortem sample of young adults.

%B Atherosclerosis %V 219 %P 958-62 %8 2011 Dec %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/22015179?dopt=Abstract %R 10.1016/j.atherosclerosis.2011.09.040 %0 Journal Article %J Nat Rev Genet %D 2010 %T Taking DNA from the dead. %A McGuire, Amy L %A Majumder, Mary A %A Halpern, Scott D %A Swindell, J S %A Yaeger, Laura V %A Gibbs, Richard A %A Wheeler, Thomas M %K Cadaver %K DNA %K Ethics, Medical %K Informed Consent %B Nat Rev Genet %V 11 %P 318 %8 2010 May %G eng %N 5 %1 https://www.ncbi.nlm.nih.gov/pubmed/20332774?dopt=Abstract %R 10.1038/nrg2792 %0 Journal Article %J Genome Res %D 2009 %T The completion of the Mammalian Gene Collection (MGC). %A Temple, Gary %A Gerhard, Daniela S %A Rasooly, Rebekah %A Feingold, Elise A %A Good, Peter J %A Robinson, Cristen %A Mandich, Allison %A Derge, Jeffrey G %A Lewis, Jeanne %A Shoaf, Debonny %A Collins, Francis S %A Jang, Wonhee %A Wagner, Lukas %A Shenmen, Carolyn M %A Misquitta, Leonie %A Schaefer, Carl F %A Buetow, Kenneth H %A Bonner, Tom I %A Yankie, Linda %A Ward, Ming %A Phan, Lon %A Astashyn, Alex %A Brown, Garth %A Farrell, Catherine %A Hart, Jennifer %A Landrum, Melissa %A Maidak, Bonnie L %A Murphy, Michael %A Murphy, Terence %A Rajput, Bhanu %A Riddick, Lillian %A Webb, David %A Weber, Janet %A Wu, Wendy %A Pruitt, Kim D %A Maglott, Donna %A Siepel, Adam %A Brejova, Brona %A Diekhans, Mark %A Harte, Rachel %A Baertsch, Robert %A Kent, Jim %A Haussler, David %A Brent, Michael %A Langton, Laura %A Comstock, Charles L G %A Stevens, Michael %A Wei, Chaochun %A van Baren, Marijke J %A Salehi-Ashtiani, Kourosh %A Murray, Ryan R %A Ghamsari, Lila %A Mello, Elizabeth %A Lin, Chenwei %A Pennacchio, Christa %A Schreiber, Kirsten %A Shapiro, Nicole %A Marsh, Amber %A Pardes, Elizabeth %A Moore, Troy %A Lebeau, Anita %A Muratet, Mike %A Simmons, Blake %A Kloske, David %A Sieja, Stephanie %A Hudson, James %A Sethupathy, Praveen %A Brownstein, Michael %A Bhat, Narayan %A Lazar, Joseph %A Jacob, Howard %A Gruber, Chris E %A Smith, Mark R %A McPherson, John %A Garcia, Angela M %A Gunaratne, Preethi H %A Wu, Jiaqian %A Muzny, Donna %A Gibbs, Richard A %A Young, Alice C %A Bouffard, Gerard G %A Blakesley, Robert W %A Mullikin, Jim %A Green, Eric D %A Dickson, Mark C %A Rodriguez, Alex C %A Grimwood, Jane %A Schmutz, Jeremy %A Myers, Richard M %A Hirst, Martin %A Zeng, Thomas %A Tse, Kane %A Moksa, Michelle %A Deng, Merinda %A Ma, Kevin %A Mah, Diana %A Pang, Johnson %A Taylor, Greg %A Chuah, Eric %A Deng, Athena %A Fichter, Keith %A Go, Anne %A Lee, Stephanie %A Wang, Jing %A Griffith, Malachi %A Morin, Ryan %A Moore, Richard A %A Mayo, Michael %A Munro, Sarah %A Wagner, Susan %A Jones, Steven J M %A Holt, Robert A %A Marra, Marco A %A Lu, Sun %A Yang, Shuwei %A Hartigan, James %A Graf, Marcus %A Wagner, Ralf %A Letovksy, Stanley %A Pulido, Jacqueline C %A Robison, Keith %A Esposito, Dominic %A Hartley, James %A Wall, Vanessa E %A Hopkins, Ralph F %A Ohara, Osamu %A Wiemann, Stefan %K Animals %K Cloning, Molecular %K Computational Biology %K DNA %K DNA, Complementary %K Gene Library %K Genes %K Humans %K Mammals %K Mice %K National Institutes of Health (U.S.) %K Rats %K Reverse Transcriptase Polymerase Chain Reaction %K United States %X

Since its start, the Mammalian Gene Collection (MGC) has sought to provide at least one full-protein-coding sequence cDNA clone for every human and mouse gene with a RefSeq transcript, and at least 6200 rat genes. The MGC cloning effort initially relied on random expressed sequence tag screening of cDNA libraries. Here, we summarize our recent progress using directed RT-PCR cloning and DNA synthesis. The MGC now contains clones with the entire protein-coding sequence for 92% of human and 89% of mouse genes with curated RefSeq (NM-accession) transcripts, and for 97% of human and 96% of mouse genes with curated RefSeq transcripts that have one or more PubMed publications, in addition to clones for more than 6300 rat genes. These high-quality MGC clones and their sequences are accessible without restriction to researchers worldwide.

%B Genome Res %V 19 %P 2324-33 %8 2009 Dec %G eng %N 12 %1 https://www.ncbi.nlm.nih.gov/pubmed/19767417?dopt=Abstract %R 10.1101/gr.095976.109 %0 Journal Article %J Anal Biochem %D 2009 %T High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil. %A Lin, Jianghai %A Kennedy, Stephen H %A Svarovsky, Therese %A Rogers, Jeffrey %A Kemnitz, Joseph W %A Xu, Anlong %A Zondervan, Krina T %K DNA %K Fixatives %K Formaldehyde %K Mineral Oil %K Paraffin Embedding %K Polymerase Chain Reaction %K Time Factors %X

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88-100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.

%B Anal Biochem %V 395 %P 265-7 %8 2009 Dec 15 %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/19698695?dopt=Abstract %R 10.1016/j.ab.2009.08.016 %0 Journal Article %J Nat Genet %D 2009 %T Personalized copy number and segmental duplication maps using next-generation sequencing. %A Alkan, Can %A Kidd, Jeffrey M %A Marques-Bonet, Tomas %A Aksay, Gozde %A Antonacci, Francesca %A Hormozdiari, Fereydoun %A Kitzman, Jacob O %A Baker, Carl %A Malig, Maika %A Mutlu, Onur %A Sahinalp, S Cenk %A Gibbs, Richard A %A Eichler, Evan E %K Algorithms %K Chromosome Mapping %K DNA %K Gene Dosage %K Gene Duplication %K Genome, Human %K Genomic Library %K Humans %K Polymorphism, Genetic %K Sequence Analysis, DNA %X

Despite their importance in gene innovation and phenotypic variation, duplicated regions have remained largely intractable owing to difficulties in accurately resolving their structure, copy number and sequence content. We present an algorithm (mrFAST) to comprehensively map next-generation sequence reads, which allows for the prediction of absolute copy-number variation of duplicated segments and genes. We examine three human genomes and experimentally validate genome-wide copy number differences. We estimate that, on average, 73-87 genes vary in copy number between any two individuals and find that these genic differences overwhelmingly correspond to segmental duplications (odds ratio = 135; P < 2.2 x 10(-16)). Our method can distinguish between different copies of highly identical genes, providing a more accurate assessment of gene content and insight into functional constraint without the limitations of array-based technology.

%B Nat Genet %V 41 %P 1061-7 %8 2009 Oct %G eng %N 10 %1 https://www.ncbi.nlm.nih.gov/pubmed/19718026?dopt=Abstract %R 10.1038/ng.437 %0 Journal Article %J Bioinformatics %D 2008 %T cneViewer: a database of conserved non-coding elements for studies of tissue-specific gene regulation. %A Persampieri, Jason %A Ritter, Deborah I %A Lees, Daniel %A Lehoczky, Jessica %A Li, Qiang %A Guo, Su %A Chuang, Jeffrey H %K Animals %K Base Sequence %K Computational Biology %K Conserved Sequence %K Databases, Genetic %K DNA %K Gene Expression Regulation %K Humans %K Internet %K Molecular Sequence Data %K Regulatory Sequences, Nucleic Acid %K Software %K Zebrafish %X

There are thousands of strongly conserved non-coding elements (CNEs) in vertebrate genomes, and their functions remain largely unknown. However, without biologically relevant criteria for prioritizing them, selecting a particular CNE sequences to study can be haphazard. To address this problem, we present cneViewer-a database and webtool that systematizes information on conserved non-coding DNA elements in zebrafish. A key feature here is the ability to search for CNEs that may be relevant to tissue-specific gene regulation, based on known developmental expression patterns of nearby genes. cneViewer provides this and other organizing features that significantly facilitate experimental design and CNE analysis.

%B Bioinformatics %V 24 %P 2418-9 %8 2008 Oct 15 %G eng %N 20 %1 https://www.ncbi.nlm.nih.gov/pubmed/18718943?dopt=Abstract %R 10.1093/bioinformatics/btn443 %0 Journal Article %J Nucleic Acids Res %D 2007 %T Termination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates. %A Wu, Weidong %A Stupi, Brian P %A Litosh, Vladislav A %A Mansouri, Dena %A Farley, Demetra %A Morris, Sidney %A Metzker, Sherry %A Metzker, Michael L %K Alkylation %K Base Pair Mismatch %K Deoxyadenine Nucleotides %K Deoxyadenosines %K DNA %K DNA-Directed DNA Polymerase %K Photochemistry %K Sequence Analysis, DNA %K Ultraviolet Rays %X

The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the 'next-generation' technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N(6)-position of 2'-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3'-OH group of the N(6)-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N(6)-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.

%B Nucleic Acids Res %V 35 %P 6339-49 %8 2007 %G eng %N 19 %1 https://www.ncbi.nlm.nih.gov/pubmed/17881370?dopt=Abstract %R 10.1093/nar/gkm689 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2006 %T Recurrent duplication-driven transposition of DNA during hominoid evolution. %A Johnson, Matthew E %A Cheng, Ze %A Morrison, V Anne %A Scherer, Steven %A Ventura, Mario %A Gibbs, Richard A %A Green, Eric D %A Eichler, Evan E %K Animals %K Base Sequence %K Biological Evolution %K Chromosomes, Human, Pair 16 %K DNA %K DNA Transposable Elements %K Evolution, Molecular %K Gene Duplication %K Hominidae %K Humans %K Molecular Sequence Data %K Phylogeny %K Sequence Alignment %K Sequence Analysis, DNA %X

The underlying mechanism by which the interspersed pattern of human segmental duplications has evolved is unknown. Based on a comparative analysis of primate genomes, we show that a particular segmental duplication (LCR16a) has been the source locus for the formation of the majority of intrachromosomal duplications blocks on human chromosome 16. We provide evidence that this particular segment has been active independently in each great ape and human lineage at different points during evolution. Euchromatic sequence that flanks sites of LCR16a integration are frequently lineage-specific duplications. This process has mobilized duplication blocks (15-200 kb in size) to new genomic locations in each species. Breakpoint analysis of lineage-specific insertions suggests coordinated deletion of repeat-rich DNA at the target site, in some cases deleting genes in that species. Our data support a model of duplication where the probability that a segment of DNA becomes duplicated is determined by its proximity to core duplicons, such as LCR16a.

%B Proc Natl Acad Sci U S A %V 103 %P 17626-31 %8 2006 Nov 21 %G eng %N 47 %1 https://www.ncbi.nlm.nih.gov/pubmed/17101969?dopt=Abstract %R 10.1073/pnas.0605426103 %0 Journal Article %J Dev Biol %D 2006 %T Shedding genomic light on Aristotle's lantern. %A Sodergren, Erica %A Shen, Yufeng %A Song, Xingzhi %A Zhang, Lan %A Richard A Gibbs %A Weinstock, George M %K Animals %K Chromosomes, Artificial, Bacterial %K DNA %K Genome %K Sea Urchins %X

Sea urchins have proved fascinating to biologists since the time of Aristotle who compared the appearance of their bony mouth structure to a lantern in The History of Animals. Throughout modern times it has been a model system for research in developmental biology. Now, the genome of the sea urchin Strongylocentrotus purpuratus is the first echinoderm genome to be sequenced. A high quality draft sequence assembly was produced using the Atlas assembler to combine whole genome shotgun sequences with sequences from a collection of BACs selected to form a minimal tiling path along the genome. A formidable challenge was presented by the high degree of heterozygosity between the two haplotypes of the selected male representative of this marine organism. This was overcome by use of the BAC tiling path backbone, in which each BAC represents a single haplotype, as well as by improvements in the Atlas software. Another innovation introduced in this project was the sequencing of pools of tiling path BACs rather than individual BAC sequencing. The Clone-Array Pooled Shotgun Strategy greatly reduced the cost and time devoted to preparing shotgun libraries from BAC clones. The genome sequence was analyzed with several gene prediction methods to produce a comprehensive gene list that was then manually refined and annotated by a volunteer team of sea urchin experts. This latter annotation community edited over 9000 gene models and uncovered many unexpected aspects of the sea urchin genetic content impacting transcriptional regulation, immunology, sensory perception, and an organism's development. Analysis of the basic deuterostome genetic complement supports the sea urchin's role as a model system for deuterostome and, by extension, chordate development.

%B Dev Biol %V 300 %P 2-8 %8 2006 Dec 01 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/17097628?dopt=Abstract %R 10.1016/j.ydbio.2006.10.005 %0 Journal Article %J J Biol Chem %D 2005 %T The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor trafficking regulator "stargazin" is related to the claudin family of proteins by Its ability to mediate cell-cell adhesion. %A Price, Maureen G %A Davis, Caleb F %A Deng, Fang %A Burgess, Daniel L %K Amino Acid Sequence %K Animals %K Base Sequence %K Calcium Channels %K Cell Adhesion %K Cell Line %K DNA %K Humans %K L Cells %K Membrane Proteins %K Mice %K Microscopy, Electron %K Molecular Sequence Data %K Multigene Family %K Mutation %K Neurons %K Phylogeny %K Receptors, AMPA %K Recombinant Proteins %K Sequence Homology, Amino Acid %K Signal Transduction %K Transfection %X

Mutations in the Cacng2 gene encoding the neuronal transmembrane protein stargazin result in recessively inherited epilepsy and ataxia in "stargazer" mice. Functional studies suggest a dual role for stargazin, both as a modulatory gamma subunit for voltage-dependent calcium channels and as a regulator of post-synaptic membrane targeting for alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors. Co-immunoprecipitation experiments demonstrate that stargazin can bind proteins of either complex in vivo, but it remains unclear whether it can associate with both complexes simultaneously. Cacng2 is one of eight closely related genes (Cacng1-8) encoding proteins with four transmembrane segments, cytoplasmic termini, and molecular masses between 25 and 44 kDa. This group of Cacng genes constitutes only one branch of a larger monophyletic assembly dominated by over 20 genes encoding proteins known as claudins. Claudins regulate cell adhesion and paracellular permeability as fundamental components of non-neuronal tight junctions. Because stargazin is structurally similar to claudins, we hypothesized that it might also have retained claudin-like functions inherited from a common ancestor. Here, we report that expression of stargazin in mouse L-fibroblasts results in cell aggregation comparable with that produced by claudins, and present evidence that the interaction is heterotypic and calcium dependent. The data suggest that the cell adhesion function of stargazin preceded its current role in neurons as a regulator of either voltage-dependent calcium channels or AMPA receptors. We speculate these complexes may have co-opted the established presence of stargazin at sites of close cell-cell contact to facilitate their own evolving intercellular signaling functions.

%B J Biol Chem %V 280 %P 19711-20 %8 2005 May 20 %G eng %N 20 %1 https://www.ncbi.nlm.nih.gov/pubmed/15760900?dopt=Abstract %R 10.1074/jbc.M500623200 %0 Journal Article %J Genome Res %D 2005 %T Emerging technologies in DNA sequencing. %A Metzker, Michael L %K Animals %K Base Sequence %K DNA %K DNA-Directed DNA Polymerase %K Humans %K Sequence Analysis, DNA %X

Demand for DNA sequence information has never been greater, yet current Sanger technology is too costly, time consuming, and labor intensive to meet this ongoing demand. Applications span numerous research interests, including sequence variation studies, comparative genomics and evolution, forensics, and diagnostic and applied therapeutics. Several emerging technologies show promise of delivering next-generation solutions for fast and affordable genome sequencing. In this review article, the DNA polymerase-dependent strategies of Sanger sequencing, single nucleotide addition, and cyclic reversible termination are discussed to highlight recent advances and potential challenges these technologies face in their development for ultrafast DNA sequencing.

%B Genome Res %V 15 %P 1767-76 %8 2005 Dec %G eng %N 12 %1 https://www.ncbi.nlm.nih.gov/pubmed/16339375?dopt=Abstract %R 10.1101/gr.3770505 %0 Journal Article %J Genome Res %D 2005 %T Pooled genomic indexing of rhesus macaque. %A Milosavljevic, Aleksandar %A Harris, Ronald A %A Sodergren, Erica J %A Jackson, Andrew R %A Kalafus, Ken J %A Hodgson, Anne %A Cree, Andrew %A Dai, Weilie %A Csuros, Miklos %A Zhu, Baoli %A De Jong, Pieter J %A Weinstock, George M %A Gibbs, Richard A %K Animals %K Chromosome Aberrations %K Chromosomes, Artificial, Bacterial %K Contig Mapping %K DNA %K Genetic Markers %K Genome %K Genome, Human %K Humans %K Macaca mulatta %K Sequence Alignment %K Sequence Analysis, DNA %X

Pooled genomic indexing (PGI) is a method for mapping collections of bacterial artificial chromosome (BAC) clones between species by using a combination of clone pooling and DNA sequencing. PGI has been used to map a total of 3858 BAC clones covering approximately 24% of the rhesus macaque (Macaca mulatta) genome onto 4178 homologous loci in the human genome. A number of intrachromosomal rearrangements were detected by mapping multiple segments within the individual rhesus BACs onto multiple disjoined loci in the human genome. Transversal pooling designs involving shuffled BAC arrays were employed for robust mapping even with modest DNA sequence read coverage. A further innovation, short-tag pooled genomic indexing (ST-PGI), was also introduced to further improve the economy of mapping by sequencing multiple, short, mapable tags within a single sequencing reaction.

%B Genome Res %V 15 %P 292-301 %8 2005 Feb %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/15687293?dopt=Abstract %R 10.1101/gr.3162505 %0 Journal Article %J Genome Res %D 2004 %T Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome. %A Zhang, Zhengdong %A Burch, Paula E %A Cooney, Austin J %A Lanz, Rainer B %A Pereira, Fred A %A Wu, Jiaqian %A Gibbs, Richard A %A Weinstock, George %A Wheeler, David A %K Amino Acid Sequence %K Animals %K Chromosome Mapping %K DNA %K Evolution, Molecular %K Exons %K Genome %K Genome, Human %K Humans %K Ligands %K Mice %K Molecular Sequence Data %K Phylogeny %K Protein Structure, Quaternary %K Protein Structure, Tertiary %K Rats %K Receptors, Androgen %K Receptors, Cytoplasmic and Nuclear %K Receptors, Estrogen %K Receptors, Glucocorticoid %K Receptors, Progesterone %X

Completion of the Rattus norvegicus genome sequence enabled a global inventory and analysis of the nuclear receptors (NRs) in three mammalian species. Forty-nine NR members were found in mouse, 48 in human. Forty-seven were found in the rat, with gaps at the locations expected for the other two. Pairwise comparisons of their distribution in rat, mouse, and human identified 11 syntenic NR gene blocks, including three small clusters of two or three closely related genes, each spanning 40 kb to 1700 kb. The exon structure of the ligand-binding domain suggests that exon shuffling has played a role in the evolution of this family. An invariant splice junction in all members of the NR family except LXRbeta suggests a functional role for the intron. The ligand-binding domains of PXR and CAR are among the most divergent in the family. Their higher nucleotide substitution rates may be related to the central role played by these two NRs in the metabolism of the foreign compounds and may have resulted from limited positive selection.

%B Genome Res %V 14 %P 580-90 %8 2004 Apr %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/15059999?dopt=Abstract %R 10.1101/gr.2160004 %0 Journal Article %J Genome Res %D 2004 %T Pash: efficient genome-scale sequence anchoring by Positional Hashing. %A Kalafus, Ken J %A Jackson, Andrew R %A Milosavljevic, Aleksandar %K Animals %K Chromosome Mapping %K Cluster Analysis %K Computational Biology %K DNA %K Genome %K Genome, Human %K Humans %K Mice %K Pan troglodytes %K Rats %K Sensitivity and Specificity %K Software %X

Pash is a computer program for efficient, parallel, all-against-all comparison of very long DNA sequences. Pash implements Positional Hashing, a novel parallelizable method for sequence comparison based on k-mer representation of sequences. The Positional Hashing method breaks the comparison problem in a unique way that avoids the quadratic penalty encountered with other sensitive methods and confers inherent low-level parallelism. Furthermore, Positional Hashing allows one to readily and predictably trade between sensitivity and speed. In a simulated comparison task, anchoring computationally mutated reads onto a genome, the sensitivity of Pash was equal to or greater than that of BLAST and BLAT, with Pash outperforming these programs as the reads became shorter and less similar to the genome. Using modest computing resources, we employed Pash for two large-scale sequence comparison tasks: comparison of three mammalian genomes, and anchoring millions of chimpanzee whole-genome shotgun sequencing reads onto the human genome. The results of these comparisons by Pash agree with those computed by other methods that use more than an order of magnitude more computing resources. These results confirm the sensitivity of Positional Hashing.

%B Genome Res %V 14 %P 672-8 %8 2004 Apr %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/15060009?dopt=Abstract %R 10.1101/gr.1963804 %0 Journal Article %J Am J Hum Genet %D 2004 %T Somatic and germline instability of the ATTCT repeat in spinocerebellar ataxia type 10. %A Matsuura, Tohru %A Fang, Ping %A Lin, Xi %A Khajavi, Mehrdad %A Tsuji, Kuniko %A Rasmussen, Astrid %A Grewal, Raji P %A Achari, Madhureeta %A Alonso, Maria E %A Pulst, Stefan M %A Zoghbi, Huda Y %A Nelson, David L %A Roa, Benjamin B %A Ashizawa, Tetsuo %K Age Distribution %K Cell Line %K Disease Transmission, Infectious %K DNA %K Female %K Genes, Dominant %K Germ Cells %K Humans %K Leukocytes %K Male %K Microsatellite Repeats %K Mouth Mucosa %K Pedigree %K Repetitive Sequences, Nucleic Acid %K Spermatozoa %K Spinocerebellar Ataxias %X

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant disorder characterized by ataxia, seizures, and anticipation. It is caused by an expanded ATTCT pentanucleotide repeat in intron 9 of a novel gene, designated "SCA10." The ATTCT expansion in SCA10 represents a novel class of microsatellite repeat and is one of the largest found to cause human diseases. The expanded ATTCT repeat is unstably transmitted from generation to generation, and an inverse correlation has been observed between size of repeat and age at onset. In this multifamily study, we investigated the intergenerational instability, somatic and germline mosaicism, and age-dependent repeat-size changes of the expanded ATTCT repeat. Our results showed that (1) the expanded ATTCT repeats are highly unstable when paternally transmitted, whereas maternal transmission resulted in significantly smaller changes in repeat size; (2) blood leukocytes, lymphoblastoid cells, buccal cells, and sperm have a variable degree of mosaicism in ATTCT expansion; (3) the length of the expanded repeat was not observed to change in individuals over a 5-year period; and (4) clinically determined anticipation is sometimes associated with intergenerational contraction rather than expansion of the ATTCT repeat.

%B Am J Hum Genet %V 74 %P 1216-24 %8 2004 Jun %G eng %N 6 %1 https://www.ncbi.nlm.nih.gov/pubmed/15127363?dopt=Abstract %R 10.1086/421526 %0 Journal Article %J Nature %D 2003 %T The International HapMap Project. %K Base Sequence %K DNA %K Gene Frequency %K Genetic Variation %K Genome, Human %K Genomics %K Haplotypes %K Humans %K International Cooperation %K Polymorphism, Single Nucleotide %K Public Sector %K Racial Groups %X

The goal of the International HapMap Project is to determine the common patterns of DNA sequence variation in the human genome and to make this information freely available in the public domain. An international consortium is developing a map of these patterns across the genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them, in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance our ability to choose targets for therapeutic intervention.

%B Nature %V 426 %P 789-96 %8 2003 Dec 18 %G eng %N 6968 %1 https://www.ncbi.nlm.nih.gov/pubmed/14685227?dopt=Abstract %R 10.1038/nature02168 %0 Journal Article %J Chemistry %D 2003 %T Rigid, conjugated, fluoresceinated thymidine triphosphates: syntheses and polymerase mediated incorporation into DNA analogues. %A Thoresen, Lars H %A Jiao, Guan-Sheng %A Haaland, Wade C %A Metzker, Michael L %A Burgess, Kevin %K DNA %K Fluoresceins %K Spectrometry, Fluorescence %K Spectrophotometry, Ultraviolet %K Substrate Specificity %K Taq Polymerase %K Thymine Nucleotides %X

Syntheses of a unique set of energy transfer dye labeled nucleoside triphosphates, compounds 1-3, are described. Attempts to prepare these compounds were only successful if the triphosphorylation reaction was performed before coupling the dye to the nucleobase, and not the other way around. Compounds were prepared as both the 2'-deoxy (a) and 2',3'-dideoxy- (b) forms. They feature progressively longer rigid conjugated linkers connecting the nucleobase and the hydroxyxanthone moiety. UV spectra of the parent nucleosides 12-14 show that as the length of the linker increases so does the absorption of the donor in the 320-330 nm region, but with relatively little red-shift of the maxima. Fluorescence spectra of the same compounds show that radiation in the 320-330 nm region results in predominant emission from the fluorescein. When the linker is irradiated at 320 nm, the only significant emission observed corresponds to the hydroxyxanthone part of the molecules at 520 nm; this corresponds to an effective Stokes' shift of 200 nm. As the absorption at 320-330 nm by the linker increases with length, so does the intensity of the fluorescein emission. A gel assay was used to gauge relative incorporation efficiencies of compounds 1-3, dTTP, ddTTP, and 6-TAMRA-ddTTP. Throughout, the thermostable polymerase TaqFS was used, as it is the one most widely applied in high throughput DNA sequencing. This assay showed that only compounds 3 were incorporated efficiently; these have the longest linkers. Of these, the 2'-deoxy nucleoside 3 a was incorporated and did not prevent the polymerase from extending the chain further. The 2',3'-dideoxy nucleoside 3 b was incorporated only about 430 times less efficiently than ddTTP under the same conditions, and caused chain termination. The implications of these studies on modified sequencing protocols are discussed.

%B Chemistry %V 9 %P 4603-10 %8 2003 Oct 06 %G eng %N 19 %1 https://www.ncbi.nlm.nih.gov/pubmed/14566865?dopt=Abstract %R 10.1002/chem.200304944 %0 Journal Article %J Genome Res %D 2003 %T Software for automated analysis of DNA fingerprinting gels. %A Fuhrmann, Daniel R %A Krzywinski, Martin I %A Chiu, Readman %A Saeedi, Parvaneh %A Schein, Jacqueline E %A Bosdet, Ian E %A Chinwalla, Asif %A Hillier, LaDeana W %A Waterston, Robert H %A McPherson, John D %A Jones, Steven J M %A Marra, Marco A %K Animals %K Cattle %K Chromosomes, Artificial, Bacterial %K DNA %K DNA Fingerprinting %K Gels %K Mice %K Models, Chemical %K Rats %K Sepharose %K Software %X

Here we describe software tools for the automated detection of DNA restriction fragments resolved on agarose fingerprinting gels. We present a mathematical model for the location and shape of the restriction fragments as a function of fragment size, with model parameters determined empirically from "marker" lanes containing molecular size standards. Automated identification of restriction fragments involves several steps, including: image preprocessing, to put the data in a form consistent with a linear model; marker lane analysis, for determination of the model parameters; and data lane analysis, a procedure for detecting restriction fragment multiplets while simultaneously determining the amplitude curve that describes restriction fragment amplitude as a function of mobility. In validation experiments conducted on fingerprinted and sequenced Bacterial Artificial Chromosome (BAC) clones, sensitivity and specificity of restriction fragment identification exceeded 96% on restriction fragments ranging in size from 600 base pairs (bp) to 30,000 bp. The integrated suite of software tools, written in MATLAB and collectively called BandLeader, is in use at the BC Cancer Agency Genome Sciences Centre (GSC) and the Washington University Genome Sequencing Center, and has been provided to the Wellcome Trust Sanger Institute and the Whitehead Institute. Employed in a production mode at the GSC, BandLeader has been used to perform automated restriction fragment identification for more than 850,000 BAC clones for mouse, rat, bovine, and poplar fingerprint mapping projects.

%B Genome Res %V 13 %P 940-53 %8 2003 May %G eng %N 5 %1 https://www.ncbi.nlm.nih.gov/pubmed/12727910?dopt=Abstract %R 10.1101/gr.904303 %0 Journal Article %J Nucleic Acids Res %D 2002 %T Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes. %A Dederich, Debra A %A Okwuonu, Geoffrey %A Garner, Toni %A Denn, Amanda %A Sutton, Angelica %A Escotto, Michael %A Martindale, Ashley %A Delgado, Oliver %A Muzny, Donna M %A Gibbs, Richard A %A Metzker, Michael L %K Animals %K DNA %K Genome %K Glass %K Microspheres %K Plasmids %K Rats %K Reproducibility of Results %K Sequence Analysis, DNA %X

To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was approximately 10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts.

%B Nucleic Acids Res %V 30 %P e32 %8 2002 Apr 01 %G eng %N 7 %1 https://www.ncbi.nlm.nih.gov/pubmed/11917038?dopt=Abstract %R 10.1093/nar/30.7.e32 %0 Journal Article %J Am J Hum Genet %D 2002 %T Intron-size constraint as a mutational mechanism in Rothmund-Thomson syndrome. %A Wang, Lisa L %A Worley, Kim %A Gannavarapu, Anu %A Chintagumpala, Murali M %A Levy, Moise L %A Plon, Sharon E %K 3T3 Cells %K Adenosine Triphosphatases %K Animals %K Base Sequence %K DNA %K DNA Helicases %K Humans %K Introns %K Mice %K Molecular Sequence Data %K Mutation %K RecQ Helicases %K Reverse Transcriptase Polymerase Chain Reaction %K RNA Splicing %K Rothmund-Thomson Syndrome %K Transfection %X

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder caused by deleterious mutations in the RECQL4 gene on chromosome 8. The RECQL4 gene structure is unusual because it contains many small introns <100 bp. We describe a proband with RTS who has a novel 11-bp intronic deletion, and we show that this mutation results in a 66-bp intron too small for proper splicing. Constraint on intron size may represent a general mutational mechanism, since human-genome analysis reveals that approximately 15% of genes have introns <100 bp and are therefore susceptible to size constraint. Thus, monitoring of intron size may allow detection of mutations missed by exon-by-exon approaches.

%B Am J Hum Genet %V 71 %P 165-7 %8 2002 Jul %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/12016592?dopt=Abstract %R 10.1086/341234 %0 Journal Article %J J Biol Chem %D 2002 %T Latency-associated nuclear antigen (LANA) cooperatively binds to two sites within the terminal repeat, and both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication. %A Garber, Alexander C %A Jianhong Hu %A Renne, Rolf %K Antigens, Viral %K Base Sequence %K Binding Sites %K Chromatin %K DNA %K DNA-Binding Proteins %K Dose-Response Relationship, Drug %K Humans %K Kinetics %K Molecular Sequence Data %K Mutation %K Nuclear Proteins %K Plasmids %K Point Mutation %K Protein Binding %K Protein Structure, Tertiary %K Transcription, Genetic %K Transfection %X

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus is a multifunctional protein with important roles in both transcriptional regulation and episomal maintenance. LANA is also a DNA-binding protein and has been shown to specifically bind to a region within the terminal repeat. Here, we have performed a detailed analysis of the DNA-binding activity of LANA and show that it binds two sites separated by 22 bp. We used electrophoretic mobility shift assay to quantitatively analyze the binding sites and determined that the K(d) of the high affinity site is 1.51 +/- 0.16 nm. Examination of the contribution of nucleotides near the ends of the site showed that the core binding site consists of 16 bp, 13 of which are conserved between both sites. Analysis of the affinity of each site alone and in tandem revealed that the binding to the second site is primarily due to cooperativity with the first site. Using deletion and point mutations, we show that both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication. In addition, we show that the ability of LANA to carry out these functions is directly proportional to its affinity for the sites in this region. The affinities, spacing, and cooperative binding between the two sites is similar to that of the Epstein-Barr virus dyad symmetry element oriP, suggesting a requirement for such an element in latent replication of these related DNA tumor viruses.

%B J Biol Chem %V 277 %P 27401-11 %8 2002 Jul 26 %G eng %N 30 %1 https://www.ncbi.nlm.nih.gov/pubmed/12015325?dopt=Abstract %R 10.1074/jbc.M203489200 %0 Journal Article %J Neuroscience %D 2001 %T Evolution tunes the excitability of individual neurons. %A Salkoff, L %A Butler, A %A Fawcett, G %A Kunkel, M %A McArdle, C %A Paz-y-Mino, G %A Nonet, M %A Walton, N %A Wang, Z W %A Yuan, A %A Wei, A %K Adaptation, Physiological %K Animals %K Base Sequence %K Biological Evolution %K Caenorhabditis %K Caenorhabditis elegans %K DNA %K Enhancer Elements, Genetic %K Gene Expression %K Molecular Sequence Data %K Multigene Family %K Neurons %K Potassium Channels %K Protein Isoforms %X

The relationship between the genome and the evolution of the nervous system may differ between an animal like C. elegans with 302 neurons, and mammals with tens of billions of neurons. Here we report that a class of nonconserved potassium channels highly expanded in C. elegans may play a special role in the evolution of its nervous system. The C. elegans genome contains an extended gene family of potassium channels whose members fall into two evolutionary divergent classes. One class constitutes an ancient conserved "set" of K+ channels with orthologues in both humans and Drosophila and a second larger class made up of rapidly evolving genes unique to C. elegans. Chief among this second class are novel potassium channels having four transmembrane domains per subunit that function as regulated leak conductances to modulate cell electrical excitability. This inventory of novel potassium channels is far larger in C. elegans than in humans or Drosophila. We found that, unlike conserved channel genes, the majority of these genes are expressed in very few cells. We also identified DNA enhancer elements associated with these genes that direct gene expression to individual neurons. We conclude that C. elegans may maintain an exceptionally large inventory of these channels (as well as ligand-gated channels) as an adaptive mechanism to "fine tune" individual neurons, making the most of its limited circuitry.

%B Neuroscience %V 103 %P 853-9 %8 2001 %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/11301195?dopt=Abstract %R 10.1016/s0306-4522(01)00079-3 %0 Journal Article %J Nat Genet %D 2000 %T Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10. %A Matsuura, T %A Yamagata, T %A Burgess, D L %A Rasmussen, A %A Grewal, R P %A Watase, K %A Khajavi, M %A McCall, A E %A Davis, C F %A Zu, L %A Achari, M %A Pulst, S M %A Alonso, E %A Noebels, J L %A Nelson, D L %A Zoghbi, H Y %A Ashizawa, T %K Animals %K Asian People %K Brain %K Chromosome Mapping %K Chromosomes, Human, Pair 22 %K DNA %K Epilepsy %K Female %K Humans %K Male %K Mexican Americans %K Mice %K Mice, Inbred C57BL %K Molecular Sequence Data %K Pedigree %K Polymorphism, Genetic %K Repetitive Sequences, Nucleic Acid %K Spinocerebellar Ataxias %K United States %K White People %X

Spinocerebellar ataxia type 10 (SCA10; MIM 603516; refs 1,2) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. The gene SCA10 maps to a 3.8-cM interval on human chromosome 22q13-qter (refs 1,2). Because several other SCA subtypes show trinucleotide repeat expansions, we examined microsatellites in this region. We found an expansion of a pentanucleotide (ATTCT) repeat in intron 9 of SCA10 in all patients in five Mexican SCA10 families. There was an inverse correlation between the expansion size, up to 22.5 kb larger than the normal allele, and the age of onset (r2=0.34, P=0.018). Analysis of 562 chromosomes from unaffected individuals of various ethnic origins (including 242 chromosomes from Mexican persons) showed a range of 10 to 22 ATTCT repeats with no evidence of expansions. Our data indicate that the new SCA10 intronic ATTCT pentanucleotide repeat in SCA10 patients is unstable and represents the largest microsatellite expansion found so far in the human genome.

%B Nat Genet %V 26 %P 191-4 %8 2000 Oct %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/11017075?dopt=Abstract %R 10.1038/79911 %0 Journal Article %J Genome Res %D 2000 %T PipMaker--a web server for aligning two genomic DNA sequences. %A Schwartz, S %A Zhang, Z %A Frazer, K A %A Smit, A %A Riemer, C %A Bouck, J %A Gibbs, R %A Hardison, R %A Miller, W %K Animals %K Base Sequence %K Caenorhabditis elegans %K Computational Biology %K DNA %K Escherichia coli %K Genes, Bacterial %K Genes, Helminth %K Genes, Protozoan %K Humans %K Internet %K Mice %K Molecular Sequence Data %K Salmonella typhimurium %K Sequence Alignment %K Software %X

PipMaker (http://bio.cse.psu.edu) is a World-Wide Web site for comparing two long DNA sequences to identify conserved segments and for producing informative, high-resolution displays of the resulting alignments. One display is a percent identity plot (pip), which shows both the position in one sequence and the degree of similarity for each aligning segment between the two sequences in a compact and easily understandable form. Positions along the horizontal axis can be labeled with features such as exons of genes and repetitive elements, and colors can be used to clarify and enhance the display. The web site also provides a plot of the locations of those segments in both species (similar to a dot plot). PipMaker is appropriate for comparing genomic sequences from any two related species, although the types of information that can be inferred (e.g., protein-coding regions and cis-regulatory elements) depend on the level of conservation and the time and divergence rate since the separation of the species. Gene regulatory elements are often detectable as similar, noncoding sequences in species that diverged as much as 100-300 million years ago, such as humans and mice, Caenorhabditis elegans and C. briggsae, or Escherichia coli and Salmonella spp. PipMaker supports analysis of unfinished or "working draft" sequences by permitting one of the two sequences to be in unoriented and unordered contigs.

%B Genome Res %V 10 %P 577-86 %8 2000 Apr %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/10779500?dopt=Abstract %R 10.1101/gr.10.4.577 %0 Journal Article %J Nucleic Acids Res %D 1999 %T An allylic/acyclic adenosine nucleoside triphosphate for termination of DNA synthesis by DNA template-dependent polymerases. %A Martinez, C I %A Thoresen, L H %A Gibbs, R A %A Burgess, K %K Adenosine Triphosphate %K Base Sequence %K Boron Compounds %K DNA %K DNA Primers %K DNA Replication %K DNA-Directed DNA Polymerase %K Fluorescent Dyes %K Kinetics %K Substrate Specificity %K Templates, Genetic %X

An allylic adenosine triphosphate analog (AATP) was tested as a substrate for commercially available DNA polymerases. All but one of the enzymes assayed incorporated AATP opposite thymidine (T) with concomitant termination of the elongation reaction. A concentration of only 1 microM was sufficient for complete termination of the polymerization reaction for a short template mediated by Ampli Taq DNA polymerase FS (Taq FS). This result suggests that AATP could be used as a 2',3'-dideoxyadenosine-5'-triphosphate (ddA) surrogate. Kinetics of incorporation revealed that AATP was 48 times less efficiently incorporated than ddA. Furthermore, AATP was used in dye-primer sequencing as a substitute for ddA.

%B Nucleic Acids Res %V 27 %P 1271-4 %8 1999 Mar 01 %G eng %N 5 %1 https://www.ncbi.nlm.nih.gov/pubmed/9973613?dopt=Abstract %R 10.1093/nar/27.5.1271 %0 Journal Article %J Bioinformatics %D 1998 %T BEAUTY-X: enhanced BLAST searches for DNA queries. %A Kim C Worley %A Culpepper, P %A Wiese, B A %A Smith, R F %K Amino Acid Sequence %K Computational Biology %K Databases, Factual %K DNA %K Molecular Sequence Data %K Proteins %K Sequence Alignment %K Software %X

UNLABELLED: BEAUTY (BLAST Enhanced Alignment Utility) is an enhanced version of the BLAST database search tool that facilitates identification of the functions of matched sequences. Three recent improvements to the BEAUTY program described here make the enhanced output (1) available for DNA queries, (2) available for searches of any protein database, and (3) more up-to-date, with periodic updates of the domain information.

AVAILABILITY: BEAUTY searches of the NCBI and EMBL non-redundant protein sequence databases are available from the BCM Search Launcher Web pages (http://gc.bcm.tmc. edu:8088/search-launcher/launcher.html). BEAUTY Post-Processing of submitted search results is available using the BCM Search Launcher Batch Client (version 2.6) (ftp://gc.bcm.tmc. edu/pub/software/search-launcher/).

SUPPLEMENTARY INFORMATION: Example figures are available at http://dot.bcm.tmc. edu:9331/papers/beautypp.html

CONTACT: (kworley,culpep)@bcm.tmc.edu

%B Bioinformatics %V 14 %P 890-1 %8 1998 %G eng %N 10 %1 https://www.ncbi.nlm.nih.gov/pubmed/9927720?dopt=Abstract %R 10.1093/bioinformatics/14.10.890 %0 Journal Article %J Biotechniques %D 1998 %T Elimination of residual natural nucleotides from 3'-O-modified-dNTP syntheses by enzymatic mop-up. %A Metzker, M L %A Raghavachari, R %A Burgess, K %A Richard A Gibbs %K Base Sequence %K Chromatography, High Pressure Liquid %K Deoxyribonucleotides %K DNA %K DNA-Directed DNA Polymerase %X

Here, we describe a novel strategy called enzymatic "Mop-Up" that efficiently removes contaminating dNTPs from reverse-phase, high-performance liquid chromatography (RP-HPLC) purified 3'-O-modified dNTP syntheses. Enzymatic mop-up takes advantage of the high selectivity of DNA polymerases for the former nucleoside triphosphates over the latter nucleotide analogs. We demonstrate the selective removal of contaminating dATP and dTTP from RP-HPLC purified 3'-O-methyl-dATP and 3'-O-(2-nitrobenzyl)-dTTP syntheses, respectively. These data highlight the importance of natural nucleotide contamination when interpreting enzymatic incorporation data and provide an alternative hypothesis for the observed property of catalytic editing of DNA polymerases. Moreover, the effective removal of natural nucleotides from 3'-O-modified analogs addresses the important issue of nucleotide read-through for stop-start DNA sequencing strategies, such as the base addition sequencing scheme (BASS).

%B Biotechniques %V 25 %P 814-7 %8 1998 Nov %G eng %N 5 %1 https://www.ncbi.nlm.nih.gov/pubmed/9821582?dopt=Abstract %R 10.2144/98255st01 %0 Journal Article %J J Med Genet %D 1998 %T Reassessment of biochemically determined Hunter syndrome carrier status by DNA testing. %A Timms, K M %A Edwards, F J %A Belmont, J W %A Yates, J R %A Gibbs, R A %K DNA %K Female %K Heterozygote %K Humans %K Iduronate Sulfatase %K Male %K Mucopolysaccharidosis II %K Pedigree %X

Deficiency of iduronate-2-sulphatase (IDS) results in the X linked recessive lysosomal storage disorder Hunter syndrome. Determination of carrier status in families affected by this disorder has been performed using a variety of enzymatic tests. None of these tests has proved to be 100% effective at identifying carriers. The aim of this study was to perform carrier testing in a family affected by the disorder, where testing was complicated by the fact that no surviving affected subjects were available for study. Direct dye primer sequencing of PCR products was used to identify mixed bases in an obligate carrier. Two mixed bases were observed within exon VIII. The first base change (T-->A) at nucleotide position 1150 results in a missense mutation (H342Q), while the second base change (G-->T) at nucleotide position 1151 results in a nonsense mutation (G343X). Four additional female family members were screened for the same mutation. Using this approach it is possible to provide unambiguous information about a subject's carrier status and, unlike biochemical testing, this approach will be equally effective when applied to families with the mild form of this disorder.

%B J Med Genet %V 35 %P 646-9 %8 1998 Aug %G eng %N 8 %1 https://www.ncbi.nlm.nih.gov/pubmed/9719370?dopt=Abstract %R 10.1136/jmg.35.8.646 %0 Journal Article %J Cancer Gene Ther %D 1996 %T Potential salmon sperm origin of the E3 region insert of the adenovirus 5 dl309 mutant. %A Marie-Claude Gingras %A Arevalo, P %A Aguilar-Cordova, E %K Adenovirus E3 Proteins %K Adenoviruses, Human %K Animals %K Avian Sarcoma Viruses %K Base Sequence %K DNA %K DNA Primers %K Genetic Vectors %K HeLa Cells %K Humans %K Male %K Molecular Sequence Data %K Plasmids %K Polymerase Chain Reaction %K Prolactin %K Proton-Translocating ATPases %K Repetitive Sequences, Nucleic Acid %K Salmon %K Sequence Homology, Nucleic Acid %K Spermatozoa %K Thymidine Kinase %K Transfection %X

The plasmid pJM17 is a commonly used adenoviral backbone derived from the dl309 mutant virus. It contains unknown sequences inserted in the E3 region during construction of the dl309 mutant. Complete description of the backbone sequence is required for interpretation of potential vector effects and for regulatory approval of a vector to be used in clinical trials. The anonymous E3 insert was sequenced and analyzed. The insert fragment is 646 base pairs (bp) long and is 100 bp shorter than the vector sequences it replaces. It interrupts the expression of the E3B 10.4K, 14.6K, and 14.7K genes, but not the E3A glycoprotein (gp) 19K gene. Sequence analysis and Southern blotting suggest that the insert might originate from salmon sperm DNA used as carrier during the construction of dl309. Transcription from the insert was not detected by Northern blot analysis of vector-transduced cells but was detected by reverse transcriptase polymerase chain reaction.

%B Cancer Gene Ther %V 3 %P 151-4 %8 1996 May-Jun %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/8725878?dopt=Abstract %0 Journal Article %J Nat Genet %D 1996 %T A recombination hotspot responsible for two inherited peripheral neuropathies is located near a mariner transposon-like element. %A Reiter, L T %A Murakami, T %A Koeuth, T %A Pentao, L %A Donna M Muzny %A Richard A Gibbs %A James R Lupski %K Amino Acid Sequence %K Base Sequence %K Charcot-Marie-Tooth Disease %K DNA %K DNA Transposable Elements %K Gene Deletion %K Humans %K Molecular Sequence Data %K Multigene Family %K Peripheral Nervous System Diseases %K Recombination, Genetic %K Repetitive Sequences, Nucleic Acid %K Restriction Mapping %K Sequence Homology, Amino Acid %X

The Charcot-Marie Tooth disease type 1A (CMT1A) duplication and hereditary neuropathy with liability to pressure palsies (HNPP) deletion are reciprocal products of an unequal crossing-over event between misaligned flanking CMT1A-REP repeats. The molecular aetiology of this apparently homologous recombination event was examined by sequencing the crossover region. Through the detection of novel junction fragments from the recombinant CMT1A-REPs in both CMT1A and HNPP patients, a 1.7-kb recombination hotspot within the approximately 30-kb CMT1A-REPs was identified. This hotspot is 98% identical between CMT1A-REPs indicating that sequence identity is not likely the sole factor involved in promoting crossover events. Sequence analysis revealed a mariner transposon-like element (MITE) near the hotspot which we hypothesize could mediate strand exchange events via cleavage by a transposase at or near the 3' end of the element.

%B Nat Genet %V 12 %P 288-97 %8 1996 Mar %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/8589720?dopt=Abstract %R 10.1038/ng0396-288 %0 Journal Article %J DNA Seq %D 1995 %T Complete sequence of a 38.4-kb human cosmid insert containing the polymorphic marker DXS455 from Xq28. %A Andersson, B %A Lu, F %A Donna M Muzny %A Warren, S T %A Richard A Gibbs %K Bacteriophage M13 %K Base Sequence %K Cosmids %K DNA %K Gene Library %K Genetic Markers %K Humans %K Minisatellite Repeats %K Molecular Sequence Data %K Repetitive Sequences, Nucleic Acid %K X Chromosome %X

The complete DNA sequence of a cosmid mapping to human Xq28 (DXS455) has been determined using a shotgun approach (genbank accession number: L31948). The cosmid insert is 38.4 kb in length, and contains several repetitive sequences, including a highly repetitive region located in a fragment previously shown to contain a polymorphic VNTR locus, as well as short di- and tri-nucleotide repeats. In addition to the previously known VNTR locus, a CA-repeat and an unusual 800-bp repetitive region have been found to be polymorphic. The repeated sequence mapping to the site of the VNTR locus spans 480 bp in the cosmid. In addition to several AT-dinucleotide stretches, this repeat has four copies of a 25 bp repeat unit and flanking sequences similar to this 25-mer. No expressed sequences have so far been identified in this cosmid.

%B DNA Seq %V 5 %P 219-23 %8 1995 %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/7626781?dopt=Abstract %R 10.3109/10425179509030969 %0 Journal Article %J Hum Mutat %D 1995 %T Detection and genetic analysis of beta-thalassemia mutations by competitive oligopriming. %A Athanassiadou, A %A Papachatzopoulou, A %A Richard A Gibbs %K Base Sequence %K beta-Thalassemia %K DNA %K DNA Mutational Analysis %K DNA Primers %K Globins %K Humans %K Molecular Sequence Data %K Oligodeoxyribonucleotides %X

A new approach for the detection of beta-thalassemia mutations has been applied, based on competitive oligonucleotide priming (COP) of in vitro DNA amplification at the mutation site. This method allows genotyping of the template DNA, through differential labeling of the allele-specific competitive oligoprimers and biotinylation of the common reverse primer. The system provides a basis for rapid, simple, and reliable detection of the numerous known beta-thalassemia mutations, revealing the precise nature of the mismatch in each case, and thereby facilitating the molecular genetic analysis of the disease.

%B Hum Mutat %V 6 %P 30-5 %8 1995 %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/7550228?dopt=Abstract %R 10.1002/humu.1380060107 %0 Journal Article %J Nat Genet %D 1995 %T Pressing ahead with human genome sequencing. %A Richard A Gibbs %K Animals %K Base Sequence %K Caenorhabditis %K DNA %K Genome, Fungal %K Genome, Human %K Human Genome Project %K Humans %K Molecular Sequence Data %K Saccharomyces cerevisiae %X

While waiting for revolutionary techniques to emerge, conventional approaches have been used to generate more than 40 million bases of DNA sequence from large scale projects involving different organisms. About eight megabases are from the human genome. Does this progress signal the time to move ahead for a massive human genomic sequencing effort, or should we bide our time, waiting for more technical developments? The answer is to move ahead now, choosing carefully those strategies that can be easily adapted to future methodologies.

%B Nat Genet %V 11 %P 121-5 %8 1995 Oct %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/7550337?dopt=Abstract %R 10.1038/ng1095-121 %0 Journal Article %J Am J Phys Anthropol %D 1995 %T When is ancient polymorphism a potential problem for molecular phylogenetics? %A Rogers, J %A Comuzzie, A G %K Animals %K Biological Evolution %K DNA %K DNA, Mitochondrial %K Humans %K Molecular Biology %K Phylogeny %K Polymorphism, Genetic %K Primates %K Sequence Homology, Nucleic Acid %K Species Specificity %B Am J Phys Anthropol %V 98 %P 216-8; discussion 224-8 %8 1995 Oct %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/8644883?dopt=Abstract %R 10.1002/ajpa.1330980212 %0 Journal Article %J Trends Biochem Sci %D 1995 %T Working on the assembly line. %A Richard A Gibbs %A Cockerill, M %K Base Sequence %K DNA %K Molecular Sequence Data %K Sequence Analysis, DNA %K Software %B Trends Biochem Sci %V 20 %P 162-3 %8 1995 Apr %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/7770918?dopt=Abstract %R 10.1016/s0968-0004(00)88995-1 %0 Journal Article %J Dev Dyn %D 1994 %T Expression of the platelet-derived growth factor beta receptor during organogenesis and tissue differentiation in the mouse embryo. %A Shinbrot, E %A Peters, K G %A Williams, L T %K Animals %K Blood Vessels %K Bone and Bones %K Bone Development %K Cell Differentiation %K DNA %K Embryo, Mammalian %K Embryonic and Fetal Development %K Immunohistochemistry %K In Situ Hybridization %K Mesoderm %K Mice %K Myocardium %K Receptors, Platelet-Derived Growth Factor %K RNA, Messenger %X

In this study we used in situ hybridization to localize expression of the platelet-derived growth factor beta (PDGF beta) receptor mRNA during organogenesis in the mouse embryo (E 9.5-16.5). Expression was first seen in periaortic mesenchyme (E 9.5-10.5). Later (E 12.5-E 16.5), the receptor was expressed in the mesenchymal component of many developing tissues and organs, particularly derivatives of the primitive gut. The expression was exceptionally high in mesenchyme directly supporting an epithelium, typical of many developing organs such as the trachea and intestine. However, as the mesenchyme differentiated into smooth muscle, PDGF beta receptor mRNA was no longer detected. The expression of the PDGF beta receptor mRNA in mesenchymal components of developing organs, along with its absence in epithelial tissues, indicates that it may play a role in mesenchymal-epithelial interactions during organ development. Somewhat unexpectedly, the PDGF beta receptor was highly expressed in the endothelium of small blood vessels and vascular structures such as the hyaloid plexus and choroid plexus. In large blood vessels, PDGF beta receptor mRNA was found in the mesenchyme surrounding the endothelium. This suggests that the PDGF beta receptor is involved in growth and development of blood vessels.

%B Dev Dyn %V 199 %P 169-75 %8 1994 Mar %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/8018985?dopt=Abstract %R 10.1002/aja.1001990302 %0 Journal Article %J Nucleic Acids Res %D 1994 %T Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates. %A Metzker, M L %A Raghavachari, R %A Stephen Richards %A Jacutin, S E %A Civitello, A %A Burgess, K %A Richard A Gibbs %K Base Sequence %K Chromatography, High Pressure Liquid %K Deoxyribonucleotides %K DNA %K DNA-Directed DNA Polymerase %K Molecular Sequence Data %K Molecular Structure %K Templates, Genetic %X

Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.

%B Nucleic Acids Res %V 22 %P 4259-67 %8 1994 Oct 11 %G eng %N 20 %1 https://www.ncbi.nlm.nih.gov/pubmed/7937154?dopt=Abstract %R 10.1093/nar/22.20.4259 %0 Journal Article %J Anal Biochem %D 1993 %T Large-scale sequencing library production: an adaptor-based strategy. %A Povinelli, C M %A Richard A Gibbs %K Base Sequence %K Biotechnology %K CD4 Antigens %K Cloning, Molecular %K DNA %K Evaluation Studies as Topic %K Gene Library %K Genetic Techniques %K Genetic Vectors %K Humans %K Molecular Sequence Data %K Sequence Analysis, DNA %K Software Design %X

A significant bottleneck in large-scale DNA sequencing projects is the creation of high-quality random libraries suitable for sequencing. We have utilized an adaptor-based strategy and developed a simple protocol to reliably generate single-insert random subclone M13 libraries yielding uniform coverage and > 10(3) clones per microgram of DNA. Of the resulting white plaques, 95% contained inserts, < 0.5% were due to adaptors with no insert, and none of the clones were found to contain extraneous DNA (0/600) or multiple inserts (0/300).

%B Anal Biochem %V 210 %P 16-26 %8 1993 Apr %G eng %N 1 %1 https://www.ncbi.nlm.nih.gov/pubmed/8489013?dopt=Abstract %R 10.1006/abio.1993.1144 %0 Journal Article %J Am J Phys Anthropol %D 1993 %T Nuclear DNA polymorphisms in a wild population of yellow baboons (Papio hamadryas cynocephalus) from Mikumi National Park, Tanzania. %A Rogers, J %A Kidd, K K %K Animals %K Antithrombin III %K Apolipoproteins B %K beta-N-Acetylhexosaminidases %K DNA %K Hexosaminidase B %K Papio %K Polymorphism, Genetic %K Polymorphism, Restriction Fragment Length %K Renin %K Tanzania %K Vimentin %X

Yellow baboons (Papio hamadryas cynocephalus) from Mikumi National Park, Tanzania were studied for polymorphisms in nuclear DNA. The study population consists of four social groups that inhabit overlapping home ranges and exchange males. As a result, these groups are considered to be members of a single interbreeding population. Human DNA clones were used as probes to screen five loci (AT3, REN, HEXB, VIM, and APOB) for restriction fragment length polymorphisms (RFLP). A total of 14 polymorphisms, at least one at each locus, was detected in a panel of 27 baboons tested using six restriction enzymes for each locus. Eleven of these RFLP systems have average heterozygosity values greater than 0.40. This initial screening demonstrates that human DNA clones can be used to detect significant numbers of informative DNA polymorphisms in single-copy nuclear genes of this species and suggests that the average proportion of nucleotides polymorphic across nuclear loci in this population may be between 1.0% and 1.3%.

%B Am J Phys Anthropol %V 90 %P 477-86 %8 1993 Apr %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/8097371?dopt=Abstract %R 10.1002/ajpa.1330900407 %0 Journal Article %J Biochem Med Metab Biol %D 1992 %T Characterization of human genomic yeast artificial chromosome inserts containing hexokinase 1 coding information on chromosome 10. %A Gelb, B D %A Kim C Worley %A Griffin, L D %A Adams, V %A Chinault, A C %A McCabe, E R %K Animals %K Base Sequence %K Chromosome Mapping %K Chromosomes, Fungal %K Chromosomes, Human, Pair 10 %K Cricetinae %K DNA %K Genome, Human %K Hexokinase %K Humans %K Isoenzymes %K Molecular Sequence Data %K Polymerase Chain Reaction %K Yeasts %X

Hexokinase 1 (HK1) is one of four mammalian HK isoenzymes and maps to human chromosome 10. Two yeast artificial chromosomes (YACs) were identified in the Washington University human YAC library using polymerase chain reaction (PCR) primers designed with knowledge of the human HK1 cDNA sequence. YAC B129B12 is 120 kb in length and maps entirely to chromosome 10. YAC A159D5 is 400 kb in length and appears to have resulted from a recombination of chromosome 10 with non-chromosome 10 material. We report these YACs as potential resources for those interested in HK1 gene organization and mapping, as well as those desiring additional genomic information and markers on chromosome 10.

%B Biochem Med Metab Biol %V 47 %P 265-9 %8 1992 Jun %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/1627358?dopt=Abstract %R 10.1016/0885-4505(92)90035-w %0 Journal Article %J Genomics %D 1992 %T A human dimorphism resulting from loss of an Alu. %A Edwards, M C %A Richard A Gibbs %K Animals %K Base Sequence %K Blotting, Southern %K CD4 Antigens %K DNA %K Genetic Linkage %K Humans %K Molecular Sequence Data %K Papio %K Polymerase Chain Reaction %K Polymorphism, Genetic %K Repetitive Sequences, Nucleic Acid %K Retroviruses, Simian %K Sequence Homology, Nucleic Acid %X

The molecular phylogeny of Alu and other repeated sequences in the human genome provides clues to events during primate evolution. A subclass of human Alu's has been previously identified as dimorphic insertions within members of the medium reiteration frequency (mer) class of repeats, reflecting the complicated sequence of insertion and radiation events leading to the current human genome structure. One dimorphic Alu is located within a previously unidentified mer family member, in the first intron of the human T4 (CD4) gene. The insertion (Alu+ allele) has a frequency of approximately 70% in Europeans and Africans and is homozygous in 20 Asian samples. Polymerase chain reaction amplification, direct DNA sequencing, and Southern analysis using oligonucleotide probes revealed that the Alu- allele was derived from the Alu+ allele by loss of part of the inserted sequence. Comparison with a tightly linked marker within the human genome and studies of baboon DNA samples revealed that the original insertion was a relatively early event in primate evolution, but that the Alu sequence loss leading to the dimorphism has occurred much more recently. Loss of Alu insertions therefore represents one mechanism for the generation of human Alu dimorphisms.

%B Genomics %V 14 %P 590-7 %8 1992 Nov %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/1330888?dopt=Abstract %R 10.1016/s0888-7543(05)80156-9 %0 Journal Article %J Hum Mol Genet %D 1992 %T Identification of a 2 base pair nonsense mutation causing a cryptic splice site in a DMD patient. %A Winnard, A V %A Jia-Hsu, Y %A Richard A Gibbs %A Mendell, J R %A Burghes, A H %K Amino Acid Sequence %K Base Sequence %K DNA %K Female %K Humans %K Molecular Sequence Data %K Muscular Dystrophies %K Mutation %K Polymerase Chain Reaction %K RNA Splicing %K RNA, Messenger %B Hum Mol Genet %V 1 %P 645-6 %8 1992 Nov %G eng %N 8 %1 https://www.ncbi.nlm.nih.gov/pubmed/1301174?dopt=Abstract %R 10.1093/hmg/1.8.645 %0 Journal Article %J Genomics %D 1992 %T Identification of new markers in Xp21 between DXS28 (C7) and DMD. %A Kim C Worley %A Towbin, J A %A Zhu, X M %A Barker, D F %A Ballabio, A %A Chamberlain, J %A Biesecker, L G %A Blethen, S L %A Brosnan, P %A Fox, J E %K Base Sequence %K Blotting, Southern %K Chromosome Mapping %K Chromosomes, Fungal %K DNA %K Gene Library %K Genetic Markers %K Genome, Human %K Humans %K Molecular Sequence Data %K Muscular Dystrophies %K Oligonucleotides %K Phenotype %K Polymerase Chain Reaction %K X Chromosome %X

Characterization of Xp21 distal to Duchenne muscular dystrophy (DMD) in the region containing the genes for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GKD) has been limited due to a paucity of probes. Two probes were localized between DXS28 (C7) and AHC, the yeast artificial chromosome insert YHX39 (DXS727) and the polymorphic phage clone QST59 (DXS319). A genomic clone, FT1 (DXS726), 3' to DMD, was also characterized. Portions of the three probes were sequenced and primer pairs were generated to amplify a sequence-tagged site within each probe. Amplification of DNA from patients confirmed the deletion results obtained by Southern blot analysis, and these three sequence-tagged sites were successfully combined for triplex PCR. In addition to facilitating molecular genetic diagnosis in Xp21, these probes can be used to identify additional YACs and other probes to further increase the genomic information and diagnostic capabilities in this region.

%B Genomics %V 13 %P 957-61 %8 1992 Aug %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/1505987?dopt=Abstract %R 10.1016/0888-7543(92)90007-f %0 Journal Article %J Environ Mol Mutagen %D 1992 %T Polymerase chain reaction-based comprehensive procedure for the analysis of the mutation spectrum at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster cells. %A Yu, Y J %A Xu, Z %A Richard A Gibbs %A Hsie, A W %K Animals %K Base Sequence %K CHO Cells %K Cricetinae %K DNA %K Exons %K Hypoxanthine Phosphoribosyltransferase %K Molecular Sequence Data %K Mutation %K Polymerase Chain Reaction %X

We have established a comprehensive procedure based on the polymerase chain reaction (PCR) to analyze the molecular spectrum of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. The procedure includes direct sequencing of PCR-amplified hprt cDNA for locating point mutations in the expressed coding sequences, multiplex PCR amplification of the hprt exons for screening large deletions, and direct sequencing of PCR-amplified hprt exons and their flanking regions for detecting intronic mutations resulting in mRNA splicing errors. Using this procedure, we have identified different types of mutations among a representative collection of spontaneous and induced HPRT-deficient Chinese hamster cell mutants. This procedure is simple, rapid, accurate, and practical for a comprehensive study of the mutation spectrum in a large number of HPRT-deficient Chinese hamster cell mutants.

%B Environ Mol Mutagen %V 19 %P 267-73 %8 1992 %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/1600952?dopt=Abstract %R 10.1002/em.2850190402 %0 Journal Article %J Genomics %D 1992 %T A transposon-like element in the deletion-prone region of the dystrophin gene. %A Pizzuti, A %A Pieretti, M %A Fenwick, R G %A Richard A Gibbs %A Caskey, C T %K Base Sequence %K Chromosome Deletion %K DNA %K DNA Mutational Analysis %K DNA Transposable Elements %K Dystrophin %K Humans %K Introns %K Male %K Molecular Sequence Data %K Muscular Dystrophies %K Restriction Mapping %K Sequence Homology, Nucleic Acid %X

The central portion of the dystrophin gene locus is a preferential site for deletions causing progressive muscular dystrophy of the Duchenne type (DMD). The nucleotide sequence of a deletion junction fragment from a DMD patient was determined, revealing that the proximal breakpoint of the deletion in intron 43 fell within the sequence of a transposon-like element. This segment, belonging to the THE-1 family of human transposable elements, is normally present in a complete form in intron 43 of the dystrophin gene. The deletion mutation was maternally transmitted and eliminated two-thirds of the THE-1 element. Analysis of DNA from additional DMD patients revealed a second deletion with the proximal breakpoint mapping within the same THE-1 element.

%B Genomics %V 13 %P 594-600 %8 1992 Jul %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/1322353?dopt=Abstract %R 10.1016/0888-7543(92)90129-g %0 Journal Article %J Genomics %D 1990 %T Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch-Nyhan families. %A Richard A Gibbs %A Nguyen, P N %A Edwards, A %A Civitello, A B %A Caskey, C T %K Base Sequence %K Chromosome Deletion %K DNA %K Exons %K Female %K Genes %K Genetic Carrier Screening %K Humans %K Hypoxanthine Phosphoribosyltransferase %K Lesch-Nyhan Syndrome %K Male %K Molecular Sequence Data %K Pedigree %K Polymerase Chain Reaction %X

The Lesch-Nyhan (LN) syndrome is a genetically lethal human neurological disease that results from mutations that inactivate the hypoxanthine phosphoribosyltransferase (HPRT) gene. The elucidation of the complete DNA sequence of the human HPRT gene locus has enabled the construction of multiple oligonucleotide primer sets for the simultaneous in vitro amplification of all nine HPRT exons. The multiplex polymerase chain reaction provides a facile assay for the detection of HPRT exon deletions and the reaction products can be analyzed by direct automated fluorescent DNA sequencing to identify subtle alterations in the gene. Alterations have been identified in the HPRT genes from 15 independent LN cases, and 10 LN family studies were performed. The sequencing method uses solid supports and is sufficiently simple and sensitive to be a favored approach for LN diagnosis. LN heterozygotes can be diagnosed without reference to the affected male. In addition, these procedures will be useful for somatic mutagenesis studies.

%B Genomics %V 7 %P 235-44 %8 1990 Jun %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/2347587?dopt=Abstract %R 10.1016/0888-7543(90)90545-6 %0 Journal Article %J Trans Assoc Am Physicians %D 1989 %T Automated DNA sequencing methods for detection and analysis of mutations: applications to the Lesch-Nyhan syndrome. %A Edwards, A %A Richard A Gibbs %A Nguyen, P N %A Ansorge, W %A Caskey, C T %K Base Sequence %K Chromosome Deletion %K DNA %K Female %K Genetic Carrier Screening %K Humans %K Hypoxanthine Phosphoribosyltransferase %K Lesch-Nyhan Syndrome %K Male %K Mutation %K Pedigree %K Polymerase Chain Reaction %K Polymorphism, Genetic %K Pregnancy %K Prenatal Diagnosis %K Repetitive Sequences, Nucleic Acid %K Restriction Mapping %B Trans Assoc Am Physicians %V 102 %P 185-94 %8 1989 %G eng %1 https://www.ncbi.nlm.nih.gov/pubmed/2638525?dopt=Abstract %0 Journal Article %J Teratog Carcinog Mutagen %D 1989 %T Deletion screening at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster cells using the polymerase chain reaction. %A Xu, Z D %A Yu, Y J %A Hsie, A W %A Caskey, C T %A Rossiter, B %A Richard A Gibbs %K Animals %K Cells, Cultured %K Chromosome Deletion %K DNA %K DNA-Directed DNA Polymerase %K Electrophoresis, Agar Gel %K Exons %K Gene Amplification %K Hypoxanthine Phosphoribosyltransferase %K Ultraviolet Rays %K X-Rays %X

We have developed a rapid screening method using the polymerase chain reaction (PCR) for detecting deletion mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. DNA was extracted from spontaneous and ultraviolet (UV) light- and X-ray-induced hprt-deficient mutants. Two primer sets were used to amplify 276 bp and 344 bp fragments containing the entire exon 3 and exon 9 coding sequence, respectively. The PCR was performed using Taq DNA polymerase for 40 cycles, and the PCR product was directly analyzed for the presence of the respective amplified DNA using electrophoresis on agarose gels stained with ethidium bromide. With this assay, we have analyzed 39 independently derived hprt-deficient mutants. Four of ten spontaneous mutants were found to have deletions in exon 9. UV light produced mutants with predominantly wild-type amplification patterns (10/14). X-ray induced mostly deletion patterns (11/15); six of these occurred only in exon 9, and five occurred in both exons 3 and 9. These observations are consistent with the classical notion that UV light induces predominantly missense mutations and X-ray produces a high proportion of deletion mutations. Deletion mutations occurred most frequently at the 3' end of the hprt gene, suggesting the possible existence of hot spots for deletions in this region. The PCR assay for deletion detection has the advantage that it can be completed in less than 4 hr without using radioisotopes. This assay should be useful for routine deletion screening.

%B Teratog Carcinog Mutagen %V 9 %P 177-87 %8 1989 %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/2570472?dopt=Abstract %R 10.1002/tcm.1770090306 %0 Journal Article %J Nucleic Acids Res %D 1989 %T Detection of single DNA base differences by competitive oligonucleotide priming. %A Richard A Gibbs %A Nguyen, P N %A Caskey, C T %K Animals %K Base Sequence %K Binding, Competitive %K DNA %K DNA, Single-Stranded %K DNA-Directed DNA Polymerase %K Gene Amplification %K Mice %K Mice, Mutant Strains %K Molecular Sequence Data %K Mutation %K Oligonucleotides %K Taq Polymerase %K Templates, Genetic %X

Synthetic DNA oligonucleotides can serve as efficient primers for DNA synthesis even when there is a single base mismatch between the primers and the corresponding DNA template. However, when the primer-template annealing is carried out with a mixture of primers and at low stringency the binding of a perfectly matched primer is strongly favored relative to a primer differing by a single base. This primer competition is observed over a range of oligonucleotide sizes from twelve to sixteen bases and with a variety of base mismatches. When coupled with the polymerase chain reaction, for the amplification of specific DNA sequences, competitive oligonucleotide priming provides a simple general strategy for the detection of single DNA base differences.

%B Nucleic Acids Res %V 17 %P 2437-48 %8 1989 Apr 11 %G eng %N 7 %1 https://www.ncbi.nlm.nih.gov/pubmed/2717399?dopt=Abstract %R 10.1093/nar/17.7.2437 %0 Journal Article %J Proc Natl Acad Sci U S A %D 1989 %T Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA. %A Gibbs, R A %A Nguyen, P N %A McBride, L J %A Koepf, S M %A Caskey, C T %K Alleles %K Autoanalysis %K Base Sequence %K DNA %K DNA-Directed DNA Polymerase %K Gene Amplification %K Humans %K Hypoxanthine Phosphoribosyltransferase %K Lesch-Nyhan Syndrome %K Mutation %K Nucleic Acid Hybridization %K Oligonucleotide Probes %K Pedigree %K RNA Splicing %K Software %X

The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. We have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. We also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.

%B Proc Natl Acad Sci U S A %V 86 %P 1919-23 %8 1989 Mar %G eng %N 6 %1 https://www.ncbi.nlm.nih.gov/pubmed/2928313?dopt=Abstract %R 10.1073/pnas.86.6.1919 %0 Journal Article %J Nucleic Acids Res %D 1988 %T Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. %A Chamberlain, J S %A Richard A Gibbs %A Ranier, J E %A Nguyen, P N %A Caskey, C T %K Base Sequence %K Chromosome Deletion %K Cloning, Molecular %K DNA %K Female %K Gene Amplification %K Humans %K Male %K Molecular Sequence Data %K Muscular Dystrophies %K Prenatal Diagnosis %X

The application of recombinant DNA technology to prenatal diagnosis of many recessively inherited X-linked diseases is complicated by a high frequency of heterogeneous, new mutations (1). Partial gene deletions account for more than 50% of Duchenne muscular dystrophy (DMD) lesions, and approximately one-third of all cases result from a new mutation (2-5). We report the isolation and DNA sequence of several deletion prone exons from the human DMD gene. We also describe a rapid method capable of detecting the majority of deletions in the DMD gene. This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus. We demonstrate the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.

%B Nucleic Acids Res %V 16 %P 11141-56 %8 1988 Dec 09 %G eng %N 23 %1 https://www.ncbi.nlm.nih.gov/pubmed/3205741?dopt=Abstract %R 10.1093/nar/16.23.11141 %0 Journal Article %J Science %D 1988 %T Expression of the murine Duchenne muscular dystrophy gene in muscle and brain. %A Chamberlain, J S %A Pearlman, J A %A Donna M Muzny %A Richard A Gibbs %A Ranier, J E %A Caskey, C T %A Reeves, A A %K Animals %K Brain %K DNA %K DNA, Recombinant %K Gene Expression Regulation %K Intellectual Disability %K Mice %K Mice, Inbred ICR %K Mice, Mutant Strains %K Muscles %K Muscular Dystrophy, Animal %K Mutation %K Nucleic Acid Hybridization %K Ribonuclease, Pancreatic %K RNA, Messenger %X

Complementary DNA clones were isolated that represent the 5' terminal 2.5 kilobases of the murine Duchenne muscular dystrophy (Dmd) messenger RNA (mRNA). Mouse Dmd mRNA was detectable in skeletal and cardiac muscle and at a level approximately 90 percent lower in brain. Dmd mRNA is also present, but at much lower than normal levels, in both the muscle and brain of three different strains of dystrophic mdx mice. The identification of Dmd mRNA in brain raises the possibility of a relation between human Duchenne muscular dystrophy (DMD) gene expression and the mental retardation found in some DMD males. These results also provide evidence that the mdx mutations are allelic variants of mouse Dmd gene mutations.

%B Science %V 239 %P 1416-8 %8 1988 Mar 18 %G eng %N 4846 %1 https://www.ncbi.nlm.nih.gov/pubmed/3347839?dopt=Abstract %R 10.1126/science.3347839 %0 Journal Article %J Science %D 1988 %T Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. %A Lee, C C %A Wu, X W %A Richard A Gibbs %A Cook, R G %A Donna M Muzny %A Caskey, C T %K Amino Acid Sequence %K Animals %K Base Sequence %K Cloning, Molecular %K DNA %K Gene Amplification %K Liver %K Mice %K Molecular Sequence Data %K Nucleic Acid Hybridization %K Swine %K Urate Oxidase %X

Urate oxidase (E.C. 1.7.3.3) catalyzes the oxidation of uric acid to allantoin in most mammals except humans and certain primates. The amino-terminal amino acid sequence for porcine urate oxidase was determined and used in a novel procedure for generating complementary DNA (cDNA) probes to this amino acid sequence. The procedure is based on the polymerase chain reaction and utilizes mixed oligonucleotide primers complementary to the reverse translation products of an amino acid sequence. This rapid and simple cDNA cloning procedure is generally applicable and requires only a partial amino acid sequence. A cDNA probe developed by this procedure was used to isolate a full-length porcine urate oxidase cDNA and to demonstrate the presence of homologous genomic sequences in humans.

%B Science %V 239 %P 1288-91 %8 1988 Mar 11 %G eng %N 4845 %1 https://www.ncbi.nlm.nih.gov/pubmed/3344434?dopt=Abstract %R 10.1126/science.3344434 %0 Journal Article %J Science %D 1987 %T The molecular basis of the sparse fur mouse mutation. %A Veres, G %A Richard A Gibbs %A Steven E Scherer %A Caskey, C T %K Amino Acid Sequence %K Animals %K Base Sequence %K Disease Models, Animal %K DNA %K Genes %K Mice %K Mice, Mutant Strains %K Mutation %K Ornithine Decarboxylase %K RNA, Messenger %X

The ornithine transcarbamylase-deficient sparse fur mouse is an excellent model to study the most common human urea cycle disorder. The mutation has been well characterized by both biochemical and enzymological methods, but its exact nature has not been revealed. A single base substitution in the complementary DNA for ornithine transcarbamylase from the sparse fur mouse has been identified by means of a combination of two recently described techniques for rapid mutational analysis. This strategy is simpler than conventional complementary DNA library construction, screening, and sequencing, which has often been used to find a new mutation. The ornithine transcarbamylase gene in the sparse fur mouse contains a C to A transversion that alters a histidine residue to an asparagine residue at amino acid 117.

%B Science %V 237 %P 415-7 %8 1987 Jul 24 %G eng %N 4813 %1 https://www.ncbi.nlm.nih.gov/pubmed/3603027?dopt=Abstract %R 10.1126/science.3603027