Unit

UNIT 15.9 Heat-Activatable Primers for Hot-Start PCR and Hot-Start One-Step RT-PCR: Endpoint and Real-Time Experiments

  1. Elena Hidalgo Ashrafi,
  2. Natasha Paul

Published Online: 1 OCT 2009

DOI: 10.1002/0471142727.mb1509s88

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Ashrafi, E. H. and Paul, N. 2009. Heat-Activatable Primers for Hot-Start PCR and Hot-Start One-Step RT-PCR: Endpoint and Real-Time Experiments. Current Protocols in Molecular Biology. 88:15.9:15.9.1–15.9.15.

Author Information

  1. TriLink BioTechnologies, Inc., San Diego, California

Publication History

  1. Published Online: 1 OCT 2009
  2. Published Print: OCT 2009

Abstract

Hot-start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes hot-start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phosphotriester groups onto the 3′ end of the primer allows for primer-based hot-start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a hot-start polymerase suitable for your application is not available. Curr. Protoc. Mol. Biol. 88:15.9.1-15.9.15. © 2009 by John Wiley & Sons, Inc.

Keywords:

  • PCR;
  • Hot Start PCR;
  • primer dimer;
  • mispriming;
  • thermal cycler;
  • DNA polymerase;
  • dNTPs;
  • multiplex;
  • fast-cycling PCR;
  • RT-PCR;
  • real-time PCR