%0 Journal Article %J Nat Genet %D 1993 %T Genomic scanning for expressed sequences in Xp21 identifies the glycerol kinase gene. %A Guo, W %A Kim C Worley %A Adams, V %A Mason, J %A Sylvester-Jackson, D %A Zhang, Y H %A Towbin, J A %A Fogt, D D %A Madu, S %A David A Wheeler %K Amino Acid Sequence %K Bacillus subtilis %K Base Sequence %K Chromosome Mapping %K Cloning, Molecular %K Cosmids %K Escherichia coli %K Gene Library %K Glycerol Kinase %K Humans %K Liver %K Molecular Sequence Data %K Oligodeoxyribonucleotides %K Polymerase Chain Reaction %K Sequence Homology, Amino Acid %K X Chromosome %X

Rapid genomic scanning methods are required to identify expressed sequences and we report an efficient, sensitive and specific approach which relies upon hybridization of an amplified, labeled cDNA library to digested cosmid DNA. We identified expressed sequences within a cosmid in the glycerol kinase (GK) "critical region" of Xp21 that had impressive similarity to prokaryotic GKs. We used this genomic sequence information to clone the human hepatic GK cDNA. Independent confirmation of the identity of this gene was obtained by functional complementation of GK deficient E. coli mutants with a construct containing the complete human X-linked GK coding sequence.

%B Nat Genet %V 4 %P 367-72 %8 1993 Aug %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/8401584?dopt=Abstract %R 10.1038/ng0893-367 %0 Journal Article %J Genomics %D 1993 %T Yeast artificial chromosome cloning in the glycerol kinase and adrenal hypoplasia congenita region of Xp21. %A Kim C Worley %A Ellison, K A %A Zhang, Y H %A Wang, D F %A Mason, J %A Roth, E J %A Adams, V %A Fogt, D D %A Zhu, X M %A Towbin, J A %K Adrenal Insufficiency %K Base Sequence %K Chromosome Mapping %K Chromosomes, Fungal %K Gene Library %K Genetic Markers %K Genome, Human %K Glycerol Kinase %K Humans %K Molecular Sequence Data %K Polymerase Chain Reaction %K X Chromosome %X

The adrenal hypoplasia congenita (AHC) and glycerol kinase (GK) loci are telomeric to the Duchenne muscular dystrophy locus in Xp21. We developed a pair of yeast artificial chromosome (YAC) contigs spanning at least 1.2 Mb and encompassing the region from the telomeric end of the Duchenne muscular dystrophy (DMD) locus to beyond YHX39 (DXS727), including the genes for AHC and GK. The centromeric contig consists of 13 YACs reaching more than 600 kb from DMD through GK. The telomeric contig group consists of 8 YACs containing more than 600 kb including the markers YHX39 (DXS727) and QST-59 (DXS319). Patient deletion breakpoints in the region of the two YAC contigs define at least eight intervals, and seven deletion breakpoints are contained within these contigs. In addition to the probes developed from YAC ends, we have mapped eight Alu-PCR probes amplified from a radiation-reduced somatic cell hybrid, two anonymous DNA probes, and one Alu-PCR product amplified from a cosmid end, for a total of 26 new markers within this region of 2 Mb or less. One YAC in the centromeric contig contains an insert encompassing the minimum interval for GK deficiency defined by patient deletion breakpoints, and this clone includes all or part of the GK gene.

%B Genomics %V 16 %P 407-16 %8 1993 May %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/8314578?dopt=Abstract %R 10.1006/geno.1993.1204 %0 Journal Article %J Pediatr Res %D 1992 %T Genotypic confirmation from the original dried blood specimens in a neonatal hemoglobinopathy screening program. %A Descartes, M %A Huang, Y %A Zhang, Y H %A McCabe, L L %A Richard A Gibbs %A Therrell, B L %A McCabe, E R %K Alleles %K Base Sequence %K Blood Specimen Collection %K DNA Mutational Analysis %K Genotype %K Hemoglobinopathies %K Hemoglobins, Abnormal %K Humans %K Infant, Newborn %K Mass Screening %K Molecular Sequence Data %X

Dried blood spots are used for newborn screening because of ease of sample collection, handling, and shipment. DNA is stable and accessible in the filter paper matrix. Genotypic confirmation using initial specimens is demonstrated for a regional screening program. Seventy-five blinded samples underwent DNA analysis after Hb electrophoresis. DNA was microextracted from a 1/2-inch semicircle (25 microL whole blood equivalent), amplified, and analyzed by four different methods. Direct amplification without microextraction and automated sequencing from microextracted DNA also was performed. All four analyses agreed for the A and S alleles in 70 of 75 specimens. Three disagreements were clarified by the other semicircle from the original sample: two were due to polymerase chain reaction contamination and one to contamination of one of four analytical tests. Two would have required analysis of a second specimen, one because of polymerase chain reaction failure and the second because the patient had S/beta-thalassemia. Direct amplification without microextraction was successful in an additional 77 of 78 specimens for analysis of the A, S, C, and E alleles. Automated direct sequencing from microextracted DNA was demonstrated for the A, S, and C alleles. Analysis of microextracted DNA from dried blood specimens for A and S alleles reduced the need for and costs of obtaining a second specimen for confirmation by 97%. Direct amplification without microextraction for analysis of A, S, and C alleles permits additional reduction in personnel time and costs. We have demonstrated that microextracted DNA is amenable to automated sequencing after asymmetric polymerase chain reaction. Direct genotypic confirmation can facilitate diagnosis and initiation of medical intervention.

%B Pediatr Res %V 31 %P 217-21 %8 1992 Mar %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/1561006?dopt=Abstract %R 10.1203/00006450-199203000-00005