High-efficiency Full-Length cDNA Cloning by Biotinylated CAP Trapper

Genomics. 1996 Nov 1;37(3):327-36. doi: 10.1006/geno.1996.0567.

Abstract

We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 x 10(7)/10 micrograms starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins
  • Base Sequence
  • Biotin
  • Brain Chemistry
  • Chromatography, Affinity
  • Cloning, Molecular / methods*
  • DNA, Complementary / genetics*
  • Gene Library*
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Mice
  • Molecular Sequence Data
  • Peptide Elongation Factor 1
  • Peptide Elongation Factors / genetics
  • RNA Caps / chemistry*
  • RNA, Messenger / genetics*
  • Ribonuclease, Pancreatic / metabolism
  • Streptavidin

Substances

  • Bacterial Proteins
  • DNA, Complementary
  • Peptide Elongation Factor 1
  • Peptide Elongation Factors
  • RNA Caps
  • RNA, Messenger
  • Biotin
  • Streptavidin
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Ribonuclease, Pancreatic

Grant support