Primer Design in PCR
Perhaps the most critical parameter for successful PCR is the design of Primers. All things being equal, a poorly designed primer can result in a PCR reaction that will not work. The primer sequence determines several things such as the length of the product, its melting temperature and ultimately the yield. A poorly designed primer can result in little or no product due to non-specific amplification and/or primer-dimer formation, which can become competitive enough to suppress product formation. This application note is provided to give rules that should be taken into account when designing primers for PCR. More comprehensive coverage of this subject can be found elswhere.
Primer selection
Several variables must be taken into account when designing PCR Primers. Among the most critical are:
- Primer length
- Melting Temperature (Tm)
- Specificity
- Complementary Primer Sequences
- G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches
- 3'-end Sequence
- Each of these critical elements will be discussed in turn.
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