Protocol

RT-PCR Protocols

Volume 630 of the series Methods in Molecular Biology pp 301-318

Date:

Hot Start PCR

  • Natasha PaulAffiliated withDepartment of Research and Development, TriLink BioTechnologies, Inc. Email author 
  • , Jonathan ShumAffiliated withDepartment of Research and Development, TriLink BioTechnologies, Inc.
  • , Tony LeAffiliated withDepartment of Research and Development, TriLink BioTechnologies, Inc.

* Final gross prices may vary according to local VAT.

Get Access

Abstract

Hot Start activation approaches are increasingly being used to improve the performance of PCR. Since the inception of Hot Start as a means of blocking DNA polymerase extension at lower temperatures, a number of approaches have been developed that target the essential reaction components such as magnesium ion, DNA polymerase, oligonucleotide primers, and dNTPs. Herein, five different Hot Start activation protocols are presented. The first method presents the use of barriers as a means of segregating key reaction components until a Hot Start activation step. The second and third protocols demonstrate Hot Start approaches to block DNA polymerase activity through the use of anti-DNA polymerase antibodies and accessory proteins, respectively. The fourth and fifth protocols utilize thermolabile chemical modifications to the oligonucleotide primers and dNTPs. The results presented demonstrate that all protocols significantly improve the specificity of traditional thermal cycling protocols.

Key words

PCR Hot Start PCR Primer dimer Mispriming Thermal cycler DNA polymerase dNTPs