Balanced-size and Long-Size Cloning of Full-Length, Cap-Trapped cDNAs Into Vectors of the Novel lambda-FLC Family Allows Enhanced Gene Discovery Rate and Functional Analysis

Genomics. 2001 Sep;77(1-2):79-90. doi: 10.1006/geno.2001.6601.

Abstract

We have developed a new class of cloning vectors: lambda-full-length cDNA (lambda-FLC) cloning vectors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, lambda-FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain "size balancers." Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre-lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacteriophage lambda / genetics*
  • Base Sequence
  • Cloning, Molecular / methods*
  • DNA, Complementary / genetics*
  • Gene Library
  • Genetic Vectors / genetics*
  • Humans
  • Molecular Sequence Data
  • Terminology as Topic

Substances

  • DNA, Complementary