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Full-length cDNA Cloning: A Workshop on Problems and Solutions was held at the Banbury Center, Cold Spring Harbor Laboratory, on March 23-25. It was sponsored by Merck Genome Research Institute, NIH National Cancer Institute, and Research Genetics, Inc., and organized by M. Bento Soares (University of Iowa) and Piero Carninci (Tsukuba Life Science Center, Japan). A complete report of the meeting, including an extensive section on strategies for constructing libraries enriched for full-length cDNAs, is on the Web.
Critical issues pertaining to synthesis and cloning of full-length cDNAs were identified and discussed throughout the meeting. Following are some topics on which attendees reached general consensus and made recommendations.
Starting RNA. In constructing full-length libraries, every effort should be made to isolate cytoplasmic mRNA from cells in culture or fresh (soft) tissues only, and the mRNA should be tested for contaminating nuclear RNA and DNA. Testing can be done in different ways; for example, PCR reactions can test for the presence of introns of a ubiquitously expressed gene. The use of total cellular (rather than cytoplasmic) RNA is not desirable due to the expected contamination with nuclear transcripts that will be particularly significant in the upper molecular-weight range.
Full-Length cDNA Library. Although a really full length cDNA should encompass all sequences from the 5' cap to poly (A) addition sites, a cDNA comprising the entire protein-coding sequence should be considered worthy of full-length sequencing at high accuracy. However, every effort should be made to obtain truly full length cDNAs so sequence information can be obtained from both 5' and 3' noncoding regions as well.
Quality Assessment of Full-Length Libraries. Applying a set of common criteria to every new full-length library generated will become increasingly important. As part of the characterization of every new library, a common set of probes representing mRNAs of 2 kb, 4 kb, 6 kb, and 8 kb should be used to hybridize Southern blots of library DNA, endonuclease restricted to release insert from cloning vector. Jim Hudson (Research Genetics) volunteered to identify a putative list of probes corresponding to mRNAs of different sizes and abundance levels that eventually could be made available through Research Genetics. Libraries that are enriched for full-length cDNAs should be accessible for sequencing even if Southern hybridization indicates suboptimal complexity. Those constructed according to some cap-selection procedures might not be very complex but could be extremely useful if significantly enriched for full-length cDNAs.
Sequencing of Random Primed Libraries to Generate Full-Length Sequence Information. There was very little overall enthusiasm for this idea because the goal is to generate full-length sequence and produce full-length clones that should be available without restrictions to academic and industrial communities.
Cloning Vector. Despite the advantages of certain lambda vectors to preferentially clone longer cDNAs, plasmids are considered advantageous given the ease of subsequent manipulation, sequence generation, and high cloning efficiencies that can be achieved via electroporation. En masse excision protocols from lambda libraries generally are not desirable because clone representation and frequencies may be altered significantly, and most participants seemed to favor cloning into plasmid vectors. Waclaw Szybalski (University of Wisconsin) argued that the use of single-copy pBAC-like vectors should be considered as far as cloned cDNA stability is concerned. The conditionally amplifiable pBAC is preferred. |
| Author: Aaron Hall |
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Author Bio:
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| This article can be searched using: social sciences, health colorado at denver & health sciences, 10 social sciences |
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