Basics of PCR

Basics of PCR Introduction to Polymerase Chain Reaction (PCR) PCR is a repeated series of reactions that is used to amplify a specified or target segment of DNA and has been applied to a variety of applications ranging from molecular cloning to genetic testing and forensic applications History At the time, the technology to synthesize short DNA oligonucleotides (primers) was readily available It was also already known that you could use DNA polymerase to extend a primer on a DNA template, because the primer provided the 3' OH that DNA polymerase needs to add the first nucleotide to the primer What Kary Mullis figured out was that by adding two DNA primers with a defined sequence to a reaction containing DNA template, DNA polymerase, and deoxynucelotidetriphosphate molecules (dNTPs), that he could copy (many times over) a specific region of DNA How it Works There are three basic steps to PCR: (1) Denaturation (2) Annealing (3) Extension/Elongation By changing the temperature at the appropriate steps, Mullis was able to first denature the double stranded DNA template into single strands, then have the primers bind (anneal) the DNA, and finally have the extension/elongation process occur There are two synthetic DNA primers used in PCR. One primer is often referred to as the forward primer and the other as the reverse primer These primers bind to opposite strands of DNA, define the area of DNA that is to be copied, and provide the free 3' OH that the DNA polymerase uses in the formation of a phosphodiester bond during addition of the next nucleotide The original template is denatured into two single strands of DNA. Denaturation occurs at a high temperature approximately 95-98 degrees Celsius. The temperature must be high enough to break the hydrogen bonds between the base pairs and keep them apart During step 2 (annealing), the temperature is typically lowered to 50-65 degrees Celsius. This allows the forward and reverse primers to anneal (bind) to the template DNA through hydrogen bonding of complementary base pairs In step 3 (extension/elongation), the temperature is raised to approximately 72 degrees Celsius, allowing DNA polymerase to add deoxynucleotides to the 3' end of the primers Target molecules are defined as a double-stranded DNA molecule that only contains the target sequence. In PCR, target molecules are not formed until after cycle 3 is complete