Primers length

Although the primers used in this technique are random sequences, there are several criteria that must be met to produce results. Williams et al found that the minimum primer length to detect amplification in ethidium bromide-stained agarose gels was 9 bases, and a minimum of 40% GC content was also required to produce amplification (50-80% GC content is generally used). These criteria were determined at an annealing temperature of 36° but also held true at annealing temperatures as low as 15°. 

Primer Length Primer lengths longer than 15 bases give a reduced score based on the expense of longer primers. The length of the primer is not critical to the reaction. 

The temperature in the annealing phase is most heavily dependent on primer length and the sequence used. A variety of computer programs are available to calculate the theoretical melting point (T) ) and secondary structure of the primers chosen. All possible calculations are only theoretical considerations. The most convenient and simplest way to calculate for a given primer is based on the GC content and is called... 

The set of primers has to be designed specifically for the sample DNA. The primer length is usually 10-30 base pairs (bp) and their complementary sequence must be unique in the template. Additionally, there should be no intra or inter primer complementarity in order to avoid the formation of primer-dimers. Ideally, the number of each base in the primer is relatively equal. Unusual sequences such as long stretches of polypurines or polypyrimidines and repetitive sequences must be avoided. The melting temperature for both primers should be similar and lie between 55 and 80 °C. 

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