Nested Primers for PCR

PCRweb is a powerful method to amplify specific sequences of DNA from a large complex mixture of DNA. For example, you can design PCR primers to amplify a single locus from an entire genome. From a single template molecule, you can produce over 1 billion copies of the PCR product very quickly. However, the capacity to amplify over one billion fold also increases the possibility of amplifying the wrong DNA sequence over one billion times. The specificity of PCR is determined by the specificity of the PCR primers. For example, if your primers bind to more than one locus (e.g. paralog or common domain), then more than one segment of DNA will be amplified. To control for these possibilities, investigators often employ nested primers to ensure specificity.

Nested PCR means that two pairs of PCR primers were used for a single locus (figure 1). The first pair amplified the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first PCR product (figure 4) and produce a second PCR product that will be shorter than the first one (figure 5). The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.

Figure 1. Nested PCR strategy. Segment of DNA with dots representing nondiscript DNA sequence of unspecified length. The double lines represent a large distance between the portion of DNA illustrated in this figure. The portions of DNA shown with four bases in a row represent PCR primer binding sites, though real primers would be longer.

Figure 2. The first pair of PCR primers (blue with arrows) bind to the outer pair of primer binding sites and amplify all the DNA in between these two sites.

Figure 3. PCR product after the first round of amiplificaiton. Notice that the bases outside the PCR primer pair are not present in the product.

Figure 4. Second pair of nested primers (red with arrows) bind to the first PCR product. The binding sites for the second pair of primers are a few bases "internal" to the first primer binding sites.

Figure 5. Final PCR product after second round of PCR. The length of the product is defined by the location of the internal primer binding sites.

When a complete genome sequence is known, it is easier to be sure you will not amplify the wrong locus but since very few of the world's genomes have been sequenced completely, nested primers will continue to be an important control for many experiments.


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