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Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product. This allows amplification for a low number of runs in the first round, limiting non-specific products.
The digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion or partition within an micro-well. Suicide PCR is typically used in paleogenetics or other studies where avoiding false positives and ensuring the specificity of the amplified fragment is the highest priority.
Second, the formerly obtained PCR products are combined together into the overlap extension PCR reaction, where the complementary overhangs bind pair-wise allowing the polymerase to extend the DNA strand. Eventually, outer primers targeting the external overhangs are used and the desired DNA product is amplified in the final PCR reaction.
By incorporating high throughput sequencing adaptors into the PCR primers, PCR products can be sequenced with massively parallel sequencing. Alternatively, and labour-intensively, PCR product can be cloned and sequenced. Nested PCR methods can be used to enhance the product for sequencing.
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The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure.
Fast COLD-PCR produces much faster results due to the shortened protocol, while Full COLD-PCR is essential for amplification of all possible mutations in the starting mixture of DNA. Two-round COLD-PCR is a modified version of Fast COLD-PCR. During the second round of Fast COLD-PCR nested primers are used.
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