The polymerase chain reaction (PCR) is an in vitro method used for the amplification of a specific DNA template to generate a large number of copies in the span of just a few hours. The development of the PCR method in 1983 by Kary Mullis replaced the old and slow cloning techniques which required days of work.
PCR Kits and Methods
Real-time PCR is a technique that monitors DNA amplification in real-time rather than at the end of the PCR run, as is the case for End-Point PCR.
PCR enables amplification of specific DNA fragments with the help of a thermocycler and is a widespread molecular biology technique.
PCR requires five reagents; DNA/RNA template, DNA polymerase, primers (forward and reverse), dNTPs and PCR buffers.
The benefit of PCR
Another benefit of the PCR method is its high sensitivity; theoretically, a single copy of the template is enough to start the reaction. The sensitivity and speed make the PCR techniques useful in both basic research and for commercial applications like in vitro diagnostics, forensics, and quality control.
The basic principles of the PCR method
To start a PCR reaction the following is needed:
A template: a DNA or RNA strand that we want to amplify.
Nucleotides (dCTP, dGTP, dATP, dGTP): the building blocks from which DNA is made.Primers: short nucleotide strands that will anneal to the position that we want to start and end the amplification.
DNA polymerase: the enzyme catalyzing the reaction.
(Accessory additives like buffers and Mg2+)
Our cDNA synthesis Troubleshoot will guide you though any initial setbacks.
The amplification process:
Denaturation: the DNA molecule is split into two single strands of DNA by heating (carried out in a PCR thermocycler)
Hybridization: During a cool-down phase, the primers anneal to the target sequences of the single stranded DNA
Elongation: Polymerase extends the DNA molecule starting at the primer sequences, and so doubles the template numbers
The process is repeated from Step 1 achieving an exponential increase in the number of templates until the substrate runs out or the product accumulation is halting the reaction.
Check out our End-Point PCR Troubleshoot if you encounter any problems.
The past, present and future of PCR
PCR was originally developed to detect specific DNA sequences in a sample, and though this is still the most used application, many other applications have been developed over the past decades.
Nowadays there exist different types of PCRs e.g. End-Point PCR, qPCR (quantitative Real-time PCR), RT-PCR (Reverse Transcription PCR), Multiplex PCR, Nested PCR and Digital PCR. Depending on the polymerases used, the PCR is featured with special properties like a hot start mechanism or proofreading activity to enable the best results.
See our Real-Time PCR Troubleshoot as well as the other troubleshooting guides for tips to perfect your experiment.
Video and Image Credits:
Video: DNA Learning Center/YouTube