Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE)

Biotechnol Lett. 2011 Jul;33(7):1301-7. doi: 10.1007/s10529-011-0580-1. Epub 2011 Mar 8.

Abstract

Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Ligases / metabolism
  • DNA Primers / genetics
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Molecular Biology / methods*
  • Nucleic Acid Amplification Techniques / methods

Substances

  • DNA Primers
  • DNA, Complementary
  • DNA-Directed DNA Polymerase
  • DNA Ligases