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Touchdown PCR (CAT#: STEM-MB-0195-WXH)

Introduction

The touchdown PCR is a method of PCR by which primers avoid amplifying nonspecific sequences. The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just above this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures polymerase chain reaction results, as the nonspecific sequences to which primers anneal in early steps of amplification will "swamp out" any specific sequences because of the exponential nature of polymerase amplification.




Principle

In short, touchdown PCR is a technique to limit non-specific amplification by starting with a high annealing temperature and slowly decreasing this over PCR cycles until the desired annealing temperature is reached.

Applications

• Offers a simple and rapid means to optimize PCRs, increasing specificity, sensitivity and yield, without the need for lengthy optimizations and/or the redesigning of primers.
• Increase the specificity and sensitivity of PCR, especially its use in clinical diagnosis.

Procedure

1. Initial denature
2. Stage 1: 10 cycles (Denature, Anneal, Extensions)
3. Stage 2: 15-20 cycles (Denature, Anneal, Extensions)
4. Final Extension