![]() ![]() ![]() ![]() The 3' ends of a primer set, which includes a plus strand primer and a minus strand primer, should not be complementary to each other, nor can the 3' end of a single primer be complementary to other sequences in the primer. The three hydrogen bonds in GC pairs help prevent breathing but also increase the melting temperature of the primers. DNA "breathing" occurs when ends do not stay annealed but fray or split apart. The 3' end of primers should contain a G or C in order to clamp the primer and prevent "breathing" of ends, increasing priming efficiency. Optimal G-C content should range between 40-60%. Primer length should be 15-30 nucleotide residues (bases). There are a few common problems that arise when designing primers: 1) self-annealing of primers resulting in formation of secondary structures such as hairpin loops ( Figure 1a) 2) primer annealing to each other, rather then the DNA template, creating primer dimers ( Figure 1b) 3) drastically different melting temperatures (T m) for each primer, making it difficult to select an annealing temperature that will allow both primers to efficiently bind to their target sequence during themal cycling ( Figure 1c) (See the sections CALCULATING MELTING TEMPERATURE (T m) and MODIFICATIONS TO CYCLING CONDITIONS for more information on T ms).īelow is a list of characteristics that should be considered when designing primers. When designing a set of primers to a specific region of DNA desired for amplification, one primer should anneal to the plus strand, which by convention is oriented in the 5' → 3' direction (also known as the sense or nontemplate strand) and the other primer should complement the minus strand, which is oriented in the 3' → 5' direction (antisense or template strand).
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