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Using the TripleMaster PCR System for Robust Amplification of ,,, GC-Rich DNA Templates

Scott Tarpinian
Eppendorf 5 Prime, Boulder, CO, USA Introduction

The polymerase chain reaction (PCR*) is a powerful and sensitive technique used by scientists to amplify large quantities of DNA. Although the reaction mix and thermal cycling conditions can be adjusted to use almost any DNA sequence as a template, amplification of templates with high GC content has proven to be problematic. One difficulty is the bond stability between base pairs in GC-rich DNA sequences. Each G-C base pair contains three hydrogen bonds and is therefore more stable than an A-T base pair that has only two hydrogen bonds. Additional problems associated with the amplification of GC-rich DNA sequences during PCR are the renaturation of the template and the formation of secondary structures within each strand before the primers have a chance to anneal to their specific template sites. These properties of GC-rich DNA sequences lead to inefficient primer-template annealing and incomplete extension by the DNA Polymerase. As a result, amplification often results in little or no yield of the expected product when using standard PCR methods. In addition, amplification may result in non-specific products derived from regions other than the target DNA sequence.1, 3, 4, 6

The TripleMaster PCR System's unique mixture of three different enzymes (Taq DNA Polymerase, 3'5'proofreading enzyme, and polymerase enhancing factor) combined with its innovative reaction buffers, overcomes these obstacles and provides scientists with an ideal solution for amplification of complex GC-rich DNA templates.5

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