Run length refers to the longest number of repetitions of a single nucleotide. For example, the primer AAAAAACCCGTCGAA has a run length of 6, because AAAAAA is the maximal length run. Primers with long runs should be avoided, because they can easily misprime. Here, mispriming refers to binding to another region than the intended binding region due to the presence of a long run. Consider the following example:
Primer | 5'-AAAAAACCCGTCGAA-3' |
Template | 3'-TTTTTTTGGGCAGCTT-5' |
Primer | 5'-AAAAAACCCGTCGAA-3' |
Template | 3'-TTTTTTTGGGCAGCTT-5' |
The primer in the first example exhibits the intended binding mode leading to perfect complementarity of the template. Due to the large number of repeats, other binding modes may be possible as well, as illustrated in the second example, where there is complementary for the six bases at the primers 5' end and many mismatches in its 3' direction. To prevent such faulty binding modes, primers should exhibit a certain sequence complexity.