Discuss the Factors That Must Be Considered When Designing Primers

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The Answer is 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.

Discuss the Factors That Must Be Considered When Designing Primers

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