%0 Journal Article %J Biotechniques %D 2004 %T Large-scale RT-PCR recovery of full-length cDNA clones. %A Wu, Jia Qian %A Garcia, Angela M %A Hulyk, Steven %A Sneed, Anna %A Kowis, Carla %A Yuan, Ye %A Steffen, David %A McPherson, John D %A Gunaratne, Preethi H %A Richard A Gibbs %K Automation %K Cloning, Molecular %K DNA, Complementary %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Messenger %K Sequence Analysis, DNA %X

Pseudogenes, alternative transcripts, noncoding RNA, and polymorphisms each add extensive complexity to the mammalian transcriptome and confound estimation of the total number of genes. Despite advanced algorithms for gene prediction and several large-scale efforts to obtain cDNA clones for all human open reading frames (ORFs), no single collection is complete. To enhance this effort, we have developed a high-throughput pipeline for reverse transcription PCR (RT-PCR) gene recovery. Most importantly, novel molecular strategies for improving RT-PCR yield of transcripts that have been difficult to isolate by other means and computational strategies for clone sequence validation have been developed and optimized. This systematic gene recovery pipeline allows both rescue of predicted human and rat genes and provides insight into the complexity of the transcriptome through comparisons with existing data sets.

%B Biotechniques %V 36 %P 690-6, 698-700 %8 2004 Apr %G eng %N 4 %1 https://www.ncbi.nlm.nih.gov/pubmed/15088387?dopt=Abstract %R 10.2144/04364DD03 %0 Journal Article %J Genome Res %D 2004 %T The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). %A Gerhard, Daniela S %A Wagner, Lukas %A Feingold, Elise A %A Shenmen, Carolyn M %A Grouse, Lynette H %A Schuler, Greg %A Klein, Steven L %A Old, Susan %A Rasooly, Rebekah %A Good, Peter %A Guyer, Mark %A Peck, Allison M %A Derge, Jeffery G %A Lipman, David %A Collins, Francis S %A Jang, Wonhee %A Sherry, Steven %A Feolo, Mike %A Misquitta, Leonie %A Lee, Eduardo %A Rotmistrovsky, Kirill %A Greenhut, Susan F %A Schaefer, Carl F %A Buetow, Kenneth %A Bonner, Tom I %A Haussler, David %A Kent, Jim %A Kiekhaus, Mark %A Furey, Terry %A Brent, Michael %A Prange, Christa %A Schreiber, Kirsten %A Shapiro, Nicole %A Bhat, Narayan K %A Hopkins, Ralph F %A Hsie, Florence %A Driscoll, Tom %A Soares, M Bento %A Casavant, Tom L %A Scheetz, Todd E %A Brown-stein, Michael J %A Usdin, Ted B %A Toshiyuki, Shiraki %A Carninci, Piero %A Piao, Yulan %A Dudekula, Dawood B %A Ko, Minoru S H %A Kawakami, Koichi %A Suzuki, Yutaka %A Sugano, Sumio %A Gruber, C E %A Smith, M R %A Simmons, Blake %A Moore, Troy %A Waterman, Richard %A Johnson, Stephen L %A Ruan, Yijun %A Wei, Chia Lin %A Mathavan, S %A Gunaratne, Preethi H %A Wu, Jiaqian %A Garcia, Angela M %A Hulyk, Stephen W %A Fuh, Edwin %A Yuan, Ye %A Sneed, Anna %A Kowis, Carla %A Hodgson, Anne %A Muzny, Donna M %A McPherson, John %A Gibbs, Richard A %A Fahey, Jessica %A Helton, Erin %A Ketteman, Mark %A Madan, Anuradha %A Rodrigues, Stephanie %A Sanchez, Amy %A Whiting, Michelle %A Madari, Anup %A Young, Alice C %A Wetherby, Keith D %A Granite, Steven J %A Kwong, Peggy N %A Brinkley, Charles P %A Pearson, Russell L %A Bouffard, Gerard G %A Blakesly, Robert W %A Green, Eric D %A Dickson, Mark C %A Rodriguez, Alex C %A Grimwood, Jane %A Schmutz, Jeremy %A Myers, Richard M %A Butterfield, Yaron S N %A Griffith, Malachi %A Griffith, Obi L %A Krzywinski, Martin I %A Liao, Nancy %A Morin, Ryan %A Palmquist, Diana %A Petrescu, Anca S %A Skalska, Ursula %A Smailus, Duane E %A Stott, Jeff M %A Schnerch, Angelique %A Schein, Jacqueline E %A Jones, Steven J M %A Holt, Robert A %A Baross, Agnes %A Marra, Marco A %A Clifton, Sandra %A Makowski, Kathryn A %A Bosak, Stephanie %A Malek, Joel %K Animals %K Cloning, Molecular %K Computational Biology %K DNA Primers %K DNA, Complementary %K Gene Library %K Humans %K Mice %K National Institutes of Health (U.S.) %K Open Reading Frames %K Rats %K United States %K Xenopus laevis %K Zebrafish %X

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

%B Genome Res %V 14 %P 2121-7 %8 2004 Oct %G eng %N 10B %1 https://www.ncbi.nlm.nih.gov/pubmed/15489334?dopt=Abstract %R 10.1101/gr.2596504