Polymerase primer-dimer

Samples that have very low target mRNA concentrations often display increased background, particularly if amplification above 35 cycles is required. The initial formation of these products can occur at low initial (ambient) temperatures, and are reduced or eliminated by the hot start approach, in which wax beads are added, melted to form a solid layer above the cDNA, and then the PCR master mix added on top. The two solutions are mixed and the PCR reaction begins after the thermocycler heats to 94°C. Background is sometimes due to the formation of primer dimers, a double-stranded PCR product consisting of the two primers and their complementary sequences. Sometimes these primer dimers contain extra sequences between the primers (14). When primer dimers form, they can be a major problem as they are very efficiently amplified and compete with amplification of the target cDNA. This is not a major problem with the primer sets described in Table 1, but can be a problem with other primer sets. With persistent problems, the hot start approach could be modified to add the cDNA, the primers, and the Taq polymerase separately. 

Hot-start PCR can also reduce the amount of primer-dimer formation by increasing the stringency of primer annealing. At lower temperatures, the primers can anneal to each other via regions of complementarity, and the DNA polymerase can extend the annealed primers to produce primer-dimer, which can often be observed as a diffuse band of approximately 50 to 100 bp on an ethidium bromide-stained gel. The formation of nonspecific amplification products and primer-dimer can compete for reagent availability with the amplification of the product desired. Therefore, hot-start PCR can improve the yield of the specific PCR products. 

Taq polymerase has no 3 -proofreading exonuclease activity. Thus it can misincor-porate bases. However, the enzyme does have a 5 -exonuclease activity and therefore will nick translate. The most serious problem generally encountered is that Taq polymerase can add an extra nontanplate-coded A to the 3 -end of DNA chains. It can also use a related activity, making a primer dimeric that may or may not contain additional uncoded residues. Once such dimeric primers are created, they are efficient substrates for further amplification. These primer dimers are a major source of artifacts in PCR. However, they are usually... 

The use of a hot-start thermostable DNA polymerase is highly recommended, so as to minimize the generation of primer dimers and other artifacts at the beginning of the PGR process. The conditions described here are optimized for use with the Phire enzyme from Thermo-Fisher. However, other hot-start enzymes may also be used. In such instances, it may be necessary to optimize annealing temperatures and extension times to promote optimal cDNA yields. 

Figure 27-20 (A) Hypothetical replisome for concurrent replication of leading and lagging strands by a dimeric polymerase associated with helicase dnaB and a primosome. Open arrows indicate directions of movement of DNA, which is forming a loop as the polymerase fills a gap to complete an Okazaki fragment. The primase will then form a new primer and a new loop. From Komberg and Baker.265 (B) Electron micrograph of the primosome bound to covalently closed ( )X174 duplex replicative form. These enzymatically synthesized duplexes invariably contain a single primosome with one or two associated small DNA loops. From A. Komberg in Hubscher and Spadari,266 pp. 9,10.

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