Extended fitness of variable region primers by a novel PCR protocol

  • Lan F
  • Umeda M
  • Inoue K
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Abstract

In an attempt to isolate the variable region genes of five monoclonal natural autoantibodies, we failed to amplify all ten variable region cDNAs using a V region primer set and following conventional PCR protocols. A novel PCR procedure was established in which the reaction began with three cycles of low temperature annealing and chain extension using 5'-primer as the only primer present, and continuing with conventional PCR cycles following addition of 3'-primer. Combined with solid-phase cDNA synthesis on oligo(dT)cellulose, this new protocol rescued all failed PCR amplifications based on a conventional protocol, thereby extending the universal utility of the V region primers. A notable increase in amplification specificity was also achieved. Such a protocol could be useful in primer design and the amplification of non-antibody genes whose sequences are not well-understood.

Author-supplied keywords

  • Antibody gene
  • Polymerase chain reaction
  • Variable region primer
  • cDNA synthesis

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Authors

  • Fenghua Lan

  • Masato Umeda

  • Keizo Inoue

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