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PCR

Hot Start PCR : An Introduction

Updated April 2011 -   Hot Start PCR allows the inhibition of polymerase activity during PCR reaction preparation. By limiting polymerase activity prior to PCR cycling, Hot Start PCR reduces non-specific amplification and increases PCR product target yield.

 Hot Start PCR is commonly performed by using included chemical modifications, wax-barrier methods, and inhibition by a taq-directed antibody.

 PCR or polymerase chain reaction uses DNA polymerases which were isolated from thermostable organisms such as the Taq enzyme, a eubacterial type I DNA polymerase, and Pfu, an archaeal type B DNA polymerase.

 PCR amplifies DNA by multiple cycles of melting DNA at high temperature, annealing primers at a lower temperature, and then allowing polymerase extension at an intermediate temperature.  Unfortunately, these thermophilic DNA polymerases show a very small but measurable polymerase activity at room temperature during assembly of the experiment.

 This in-efficient DNA polymerase activity will catalyze the extension of any annealed 3' ends. After PCR amplification, the resulting product contains a mixture of specific and non-specific bands. Moreover, the 5'-3' exonuclease activity of the DNA polymerasel degrades any free 5' ends of partially annealed nucleic acids, destroying the primer and template substrates of the polymerase reaction. This results in a lower yield of the desired pcr product.

Hot Start PCR : Publications

Characterization of Family IV UDG from Aeropyrum pernix and Its Application i...

Characterization of Family IV UDG from Aeropyrum pernix and Its Application in Hot-Start PCR by Family B DNA Polymerase.

PLoS One. 2011;6(11):e27248

Authors: Liu XP, Liu JH

Abstract
Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55-65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C?U/G>U/T?U/AP?U/->U/U?U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.

PMID: 22087273 [PubMed - as supplied by publisher]

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