RecA-mediated multistrand formation for cloning PCR products into vectors: simplified process for 5'-rapid amplification of cDNA ends

Anal Biochem. 2005 Jun 1;341(1):141-7. doi: 10.1016/j.ab.2005.03.016.

Abstract

I have developed a novel rapid amplification of cDNA ends (RACE) technology that uses multistranded DNA formation mediated by the RecA protein. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary single-stranded DNA in the presence of RecA and exonuclease I. The possibility of applying this finding to the direct cloning of a 5'-RACE product onto a cDNA fragment, which does not require the use of restriction endonucleases, was explored. The results show that the terminal multistranded structure formed by the RecA-mediated reaction can be applied to RACE systems. Modifications to the RACE protocol to improve the effectiveness of the technique are also suggested.

MeSH terms

  • Base Pair Mismatch
  • Cloning, Molecular* / methods
  • DNA, Complementary / chemistry
  • DNA, Complementary / metabolism*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism
  • Genetic Vectors
  • Plasmids
  • Random Amplified Polymorphic DNA Technique / methods
  • Rec A Recombinases / chemistry*
  • Rec A Recombinases / metabolism
  • Transformation, Bacterial

Substances

  • DNA, Complementary
  • Escherichia coli Proteins
  • Rec A Recombinases