PCR Primer Designfrom Edwards and Logan 2009, "Performing Real-time PCR" Chapter 6 in Real-Time PCR: Current Technology and Applications. Good primer design is vital tool for all PCR and real-time qPCR procedures. Many PCR software programs are available for PCR primer design and other important PCR applications.
In any PCR or real-time qPCR the following criteria should be optimised: buffer composition, cycle conditions, magnesium chloride (MgCl2) concentration, primer concentration, probe concentration and template concentration. Commercial master mixes, which are widely available, simplify the optimisation and are convenient. Primers should be designed with the aid of appropriate software and attention should be paid to the optimal size of the amplicon produced in the real-time application. Real-time PCR products are usually <500 bp, this may be much smaller than the amplicon size generated on a block-based thermal cycler. The following guidelines should be observed for optimal and accurate primer design:
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- The primer 3' ends should be free from secondary structure, repetitive sequences, palindromes and highly degenerate sequences.
- Forward and reverse primers should not have significant complementary sequences.
- The forward and reverse primers should have equal GC contents, ideally between 40-70%.
- The binding sites should not have extensive secondary structure.
The CT (cycle threshold) value of a real-time PCR procedure is the number of cycles required for the fluorescent signal to exceed the background level i.e. "cross the threshold". CT values are dependent on various factors including primer design and DNA template concentration. This value is often referred to as crossing point (CP) in LightCycler terminology.
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