In this issue... 

1997 Santa Fe Highlights 
Patrinos Address 
Joint Genome Institute (JGI) Comes of Age 
JGI Sequencing 
JGI Informatics 
JGI and Bermuda Quality Sequence 
Grants Awarded for JGI Collaboration 
JGI Sequencing Clones 
Sequencing Strategies,Tools 
Gene-Discovery Resources 
Sequencing at NIH NHGRI 
Functional Genomics 
Data Surge Challenges Informaticists 
Genome Annotation: Informatics Advances Needed for Age of Functional Genomics 
ELSI: Rapid Progress Accelerates Societal Impact of Genome Research 
1999 DOE HGP Meeting Set for California 

Human Genome Project Administration 
New 5-Year Goals, Project Midpoint 
DOE, NIH Discuss Informatics 
JASON Group Review 
BER Genome Instrumentation Research 

In the News 
Private-Sector Sequencing Plan 
Bang for the Buck: Government-Backed Research Underpins Potentially High Payoff Ventures 
Palmisano Joins DOE OBER 
DNA Files series to be on NPR 
HUGO Addresses Sample Collection 
Sickle Cell Mice May Lead to New Treatments 
TIGR Sequencing 6 More Microbes 
Tuberculosis Microbe Sequenced 
C. Elegans Sequencing Nears Finish 
HGMIS Website Restructured 
cDNA Cloning Workshop Identifies Critical Issues 
Survey Identifies Growing Need for Synchrotron Analyses 
NCGR Announcements 

Publications 
Report on Functional Consequences of Gene Expression 
Book on Tuskegee Conference 
Book Focuses on Biomarker Implications, Conference Proceedings 
Genome Analysis Protocol Handbook 

Software and the Internet 
Mouse Genome Informatics Release 2.0 
New System Identifies Polymorphisms 
DOE Supports Web Site for 1997 AAAS Genome Symposium 
Expressed Human Genome Database 

Funding 
DOE ELSI 
NIH NHGRI 
NHGRI Initiates Mailing List 
U.S. Genome Research Funding 

Meeting Calendars & Acronyms 
Genome and Biotechnology Meetings 
Training Courses and Workshops 
Acronyms 

HGN archives and subscriptions   
HGP Information home 

cDNA Cloning Workshop Identifies Critical Issues

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 (http://www.mgri.org, click on "Special Reports").

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.

The next cDNA meeting is planned for March 1999 in Japan. [Bento Soares, bento-soares@uiowa.edu]

The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v9n3).