Discuss the factors that must be considered when designing primers for a PCR reaction

Question

What will be an ideal response

Answer 1

Primers must be long enough (15–30 bases) to minimize nonspecific annealing to DNA sequences other than the
target. Since guanine and cytosine bind with three hydrogen bonds and adenine and thymine bind with two
hydrogen bonds, if the ratio of GC nucleotides to AT nucleotides in the two primers is significantly different, then
one primer may bind better than the other, and the amplicon may form asymmetrically. The exact nucleotide
sequence of the primers is also important to know so that appropriate annealing temperatures can be used as
oligonucleotides with high GC content anneal at higher temperatures than those with low GC content. If the
primers have complementary sequences to each other, then "primer dimers" will form, and if a primer has
complementary sequences within the primer itself, it can become folded in a "hairpin loop." Both primer dimers
and looped primers function poorly, if at all.