Primer Hybridization

Primer Hybridization

Primers are short artificial DNA strands that are used in genetic methods such as DNA sequencing, hybridization probes, and Polymerase Chain Reaction (PCR). The actual construction of primers starts with 3'-hydroxyl nucleosides attached to a so-called Controlled-Pore Glass (CPG). The 5'-hydroxyl of the nucleosides is covered dimethoxytrityl (DMT), which prevents the building of a nucleotide chain. To add a nucleotide, DMT is chemically removed removed, and the nucleotide is added. The 5'-hydroxyl of the new nucleotide is blocked by DMT, preventing the addition of more than one nucleotide to each chain.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.

PCR Optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions. Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups.