Primers with 5' flaps improve real-time PCR

Biotechniques. 2007 Dec;43(6):770, 772, 774. doi: 10.2144/000112631.

Abstract

Primers that contain portions noncomplementary to the target region are usually used to add to the PCR product a utility sequence such as a restriction site or a universal probe binding site. We have demonstrated that primers with short 5'AT-rich overhangs increase real-time PCR fluorescent signal. The improvement is particularly significant for difficult to amplify templates, such as highly variable viral sequences or bisulfite-treated DNA.

MeSH terms

  • 5' Flanking Region / genetics*
  • DNA Primers / genetics*
  • DNA, Viral / genetics
  • Fluorescent Dyes
  • Humans
  • Reproducibility of Results
  • Restriction Mapping / methods
  • Restriction Mapping / standards
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards*
  • Sulfites

Substances

  • DNA Primers
  • DNA, Viral
  • Fluorescent Dyes
  • Sulfites
  • hydrogen sulfite