Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)

doi:https://doi.org/10.1093/nar/gni184

Title: Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)

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Abstract

The production of comprehensive cDNA clone collections is an important goal of the human and model organism genome projects. cDNA sequences are used to determine the structures of transcripts, including splice junctions, polyadenylation sites, and 5' and 3' untranslated regions (UTRs). cDNA collections are also valuable resources for functional studies of genes and proteins. Expressed Sequence Tag (EST)sequencing is the method of choice for recovering cDNAs representing a majority of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a library at random, so it realizes diminishing returns as the project progresses. To drive cDNA collections toward completion new methods are needed to recover cDNAs representing specific genes and alternative transcripts, including transcripts with low expression levels. We describe a simple and effective inverse-PCR-based method for screening plasmid libraries to recover intact cDNAs for specific transcripts. We tested the method by screening libraries used in our Drosophila EST projects for 153 transcription factor genes that were not yet represented by full-length cDNAs. We recovered target-specific clones for 104 of the genes: 46 exactly match, 30 improve and 28partially match current gene annotations. Successful application of the screening method depends on cDNA library complexity and quality of the genemore »« less

Authors:: Hoskins, Roger A; Stapleton, Mark; George, Reed A; Yu, Charles; Wan, Kenneth H; Carlson, Joseph W; Celniker, Susan E

Publication Date:: 2005-04-22

Research Org.:: Ernest Orlando Lawrence Berkeley NationalLaboratory, Berkeley, CA (US)

Sponsoring Org.:: USDOE; National Institutes of Health Grants HG002673 andL0001

OSTI Identifier:: 862165

Report Number(s):: LBNL-57496
Journal ID: ISSN 0305-1048; NARHAD; R&D Project: L0001; BnR: 400412000; TRN: US200602%%75

DOE Contract Number:: DE-AC02-05CH11231; NIHL0001

Resource Type:: Journal Article

Journal Name:: Nucleic Acids Research

Additional Journal Information:: Journal Volume: 33; Journal Issue: 21; Related Information: Journal Publication Date: 2005; Journal ID: ISSN 0305-1048

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; DROSOPHILA; FUNCTIONALS; GENES; PLASMIDS; PRODUCTION; PROTEINS; SLIP; TRANSCRIPTION FACTORS; cDNA transcriptome EST

Citation Formats


                    Hoskins, Roger A, Stapleton, Mark, George, Reed A, Yu, Charles, Wan, Kenneth H, Carlson, Joseph W, and Celniker, Susan E. Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP).  United States: N. p., 2005. 
        Web.  doi:10.1093/nar/gni184.

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                    Hoskins, Roger A, Stapleton, Mark, George, Reed A, Yu, Charles, Wan, Kenneth H, Carlson, Joseph W, & Celniker, Susan E. Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP).  United States.  https://doi.org/10.1093/nar/gni184

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                    Hoskins, Roger A, Stapleton, Mark, George, Reed A, Yu, Charles, Wan, Kenneth H, Carlson, Joseph W, and Celniker, Susan E. Fri .  
        "Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)".  United States.  https://doi.org/10.1093/nar/gni184.  https://www.osti.gov/servlets/purl/862165.

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@article{osti_862165,

  title        = {Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)},

  author       = {Hoskins, Roger A and Stapleton, Mark and George, Reed A and Yu, Charles and Wan, Kenneth H and Carlson, Joseph W and Celniker, Susan E},

  abstractNote = {The production of comprehensive cDNA clone collections is an important goal of the human and model organism genome projects. cDNA sequences are used to determine the structures of transcripts, including splice junctions, polyadenylation sites, and 5' and 3' untranslated regions (UTRs). cDNA collections are also valuable resources for functional studies of genes and proteins. Expressed Sequence Tag (EST)sequencing is the method of choice for recovering cDNAs representing a majority of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a library at random, so it realizes diminishing returns as the project progresses. To drive cDNA collections toward completion new methods are needed to recover cDNAs representing specific genes and alternative transcripts, including transcripts with low expression levels. We describe a simple and effective inverse-PCR-based method for screening plasmid libraries to recover intact cDNAs for specific transcripts. We tested the method by screening libraries used in our Drosophila EST projects for 153 transcription factor genes that were not yet represented by full-length cDNAs. We recovered target-specific clones for 104 of the genes: 46 exactly match, 30 improve and 28partially match current gene annotations. Successful application of the screening method depends on cDNA library complexity and quality of the gene models. The approach should be effective for improving cDNA collections for other model organisms and the human. It also provides a simple and rapid method for isolating cDNAs of interest in any system for which plasmid cDNA libraries and complete or partial gene sequences are available.},

  doi          = {10.1093/nar/gni184},

  url          = {https://www.osti.gov/biblio/862165},
  journal      = {Nucleic Acids Research},

  issn         = {0305-1048},

  number       = 21,

  volume       = 33,

  place        = {United States},

  year         = {2005},

  month        = {4}

}

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