PCR, A Surge Forward

What is it?

PCR or polymerase chain reaction is a technique used in molecular biology and diagnostics to amplify a single or few copies of a sequence of DNA or RNA into thousands to millions. Developed in 1983 by Kary Mullis, who also went on to win the Nobel Prize in 1993.

It changed the way for a lot of different domains. PCR is an indispensable technique used in clinical and research laboratories with a broad span of applications. PCR is used for DNA cloning, gene cloning and manipulation, gene mutagenesis and functional analysis of genes for diagnostic or monitoring purposes.

PCR is dependent on thermal cycling; that is exposing the reactants to cycles of repeated heating and cooling which allows different temperature dependent reactions to take hold.

So, how does it work?

The basic PCR set-up requires several specific components and reagents. There needs to be a DNA template that contains the DNA target sequence that is targeted for amplification. DNA polymerase is needed, more specifically taq polymerase as it is heat-resistant. Taq polymerase is an enzyme that is isolated from the thermophilic bacterium Thermus aquaticus. It can survive the high-temperature DNA denaturation phase. Primers that are complementary to the 3′ ends of each sense and anti-sense strands of the DNA target. Primers are specific and complementary to the target sequence and are often selected beforehand. More than likely the primers are artificially synthesized from a commercial biochemical supplier. Deoxynucleoside triphosphates of dNTPs are the building blocks from which taq polymerase synthesizes a new strand.

There are three steps to PCR; The first step, denaturation is the first step in the natural cycle of events and consists of heating the reaction to 94-98 degrees Celsius for 20-30 seconds. This causes DNA denaturation of the dsDNA template by breaking the hydrogen bonds between the complementary base-pairs. The result is two, ssDNA molecules. Annealing is the second step in PCR and the temperature is lowered to 50-65 degrees Celsius (122-149 F) for 20-40 seconds. This allows annealing of the primers to each of the ssDNA molecules. The temperature in the annealing step is critical because you must select a temperature low enough to allow hybridization of the primer to the strands, but high enough for the hybridization to be specific to the target sequence selected to amplify. The primer should only bind to the complementary part of the strand and no where else. If the temperature is too low the primer may bind improperly and cause issues or may not bind at all. Extension and elongation is the final step in PCR where the DNA taq polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture in a 5′ to 3′ direction. The reaction is raised to about 72 degrees Celsius. The 5′-phosphate group is condensed to the 3′-hydroxyl group at the end of the nascent or elongating new DNA strand.

Polymerase_chain_reaction

At the elongation step in each cycle, the number of DNA copies is doubled. Denaturation, annealing, and elongation constitute a single cycle.

PCR can fail for various reasons. PCR is very sensitive to contamination causing DNA amplification of erroneous DNA products. Primer-design techniques are important to improving PCR product yield and in avoiding the production of wrong DNA products. There are multiple primer rules that should be followed; Primers should be between 22-26 bases in length with optimization at 24. Specificity and Tm (Melting temperature) should be between 58-66 degrees Celsius. Both primers should have a similar Tm (+/- 2 degrees). Keep the G-C base pair content between 40-60% , optimization at 50% if possible. Avoid repetitive sequences (AAAA, TATATA) as they can cause mis-priming. Because annealing of the primer is most critical at its 3′ end, a primer that has a high G-C content at its 3′ end is more likely to cause mis-priming.

Some notable analogs of PCR are RT-PCR (Reverse transcriptase PCR) and qPCR (quantitative PCR). RT-PCR is used for amplifying DNA from RNA. Reverse transcriptase enzyme transcribes RNA template into cDNA, which is then amplified. This is widely used for gene expression profiling or to identify the sequence of an RNA transcript. This determines the expression profile of a gene if known. It can be used to map the exons and introns in the gene. qPCR is used to measure the quantity of the target sequence. It quantitatively measures starting amounts of DNA, cDNA, or RNA and measures the amount of copies in the sample. qPCR is highly specific and precise. Usually qPCR methods use fluorescent dyes, most commonly Sybr green or Taqman which measures the amount of amplified product in real time.

PCR and its PCR derivatives have evolved and changed the way clinical labs and diagnostics are performed. There is a wide range of use in medicine.

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