Uniquely designed primers will lead to generation of specific amplicons. The length of the primers is usually 18-30 bases, because the random combination of this size of a primer will hit less than once per total genomic sequence (3 billion base pairs).04
- There is a 1/4 chance (4-1) of finding an A, G, C or T in any given DNA sequence.
- There is a 1/16 chance (4-2) of finding any di-nucleotide sequence (e.g. AG).
- There is a 1/256 chance (4-4) of finding a given 4-base sequence (e.g. AGCT).
So, a seventeen base sequence will statistically be present only once in every 417 bases or approximately 17 billion.
A 17-mer or longer primer should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. Primers of this length generally are unique sequences in the human genome; however, it is important to ensure that portions of the primer do not have sequence or cross-homology with the target. Computer programs such as BLAST can be used to find regions of local similarity between sequences. The program compares nucleotide sequences to sequence databases and calculates the statistical significance of matches.
Primers longer than 30 bases do not demonstrate higher specificity. Additionally, long amplicons are more likely to cross-hybridize with other primers and sequences in the reaction mixture, and this can terminate the DNA polymerization.09
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