The discovery that certain DNA polymerase can catalyze repeated rounds of DNA synthesis from a known template in a controlled manner led to the development of the polymerase chain reaction (PCR) technique. PCR is a relatively simple technique by which a DNA or cDNA (complementary DNA) template is amplified many thousand- or million-fold quickly and reliably, generating sufficient material for subsequent analysis. PCR DNA) amplification process occurs inside a piece of equipment known as thermal cycler or PCR machine.
PCR revolutionized clinical/laboratory medicine and molecular biology in the 1980s, giving researchers a tool for analyzing DNA available only in minute quantities. The PCR process is exquisitely sensitive and requires little starting material, making PCR extremely useful, not only in basic research, but also for applications such as genetic identity testing, forensic analysis, industrial quality control and in vitro diagnostics. An important point to always note when preparing PCR amplifications, is to make a master mix of your buffer, primers and enzyme to reduce pipetting errors and control variability that may arise during the PCR (DNA) amplification process.
The basic components of a PCR cycle include:
- Template denaturation – which is responsible for the separation of target DNA strands by heating to 94oC or a higher temperature.
- Primer annealing – in which the temperature of the thermocycler is reduced to 40-60oC in order to allow for the pairing of oligos (i.e., the oligonucleotide primers) with their target sequences.
- Primer extension – in which new strand synthesis occurs at the optimal temperature (e.g., 68-70oC) for the Taq polymerase used in the amplification process.
There are various types of PCR processes, and they include:
- Basic endpoint PCR
- Hot-start PCR
- Long PCR
BASIC ENDPOINT PCR
The PCR process was originally developed to amplify short segments of a longer DNA molecule. A typical amplification reaction includes target DNA, a thermostable DNA polymerase or Taq polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium. Once assembled, the reaction is placed in a thermal cycler and subjected to a series of different temperatures for set amounts of time. This series of temperature and time adjustments is referred to as one PCR cycle. Each PCR cycle theoretically doubles the amount of targetedsequence (amplicon) in the reaction. Therefore, ten cycles should multiply the amplicon by a factor of about one thousand; 20 cycles, by a factor ofmore than a million. The number of copies of your DNA template increases exponentially.
Each step of the cycle should be optimized for each target, primer set and enzyme/buffer combination. If the temperature during the annealing and extension steps are similar, these two steps can be combined into a single step in which both primer annealing and extension take place. The amplification process is typically completed in 20-40 cycles, after which the amplified product may be analyzed for size, quantity or sequence, or it may be used directly in other procedures or applications.
Typical reactions with Taq DNA polymerase
|Component||Volume or final concentration|
|Nuclease-free water||to 20µl final volume|
|Reaction buffer (10X or 50X)||1X|
|Taq DNA polymerase||1.25u|
|Downstream primer||1µM (50pmol)|
|Upstream primer||1µM (50pmol)|
Some reaction buffers contain Mg2+, and additional MgCl2 may not be required. The optimal Mg2+ concentration depends on the template but is typically in the range of 0.5-4mM.
Assemble the reactions on ice in the order listed in the Table above. Be sure to vortex the MgCl2 solution, primers, dNTPs and reaction buffer before adding each to the reaction. When using a thermal cycler without a heated lid, overlay the reaction with 1 or 2 drops of mineral oil to prevent evaporation.
At lower temperatures, PCR primers can anneal to template sequences that are not perfectly complementary, and nonspecific amplifications can result from the residual activity that DNA polymerases have at these temperatures. To reduce this nonspecific amplification, a “hot start” can be employed to prevent undesired amplification events. Hot-start PCR also can reduce the amount of primer-dimer synthesized by increasing the stringency of primer annealing.
At lower temperatures, PCR primers can anneal to each other at regions of complementarity, and the DNA polymerase can extend annealed primers to produce primer dimer, which can appear as a diffuse band of approximately 50-100bp when visualized on a gel. The formation of nonspecific products and primer dimer can compete for reagent availability in the reaction and result in reduced amplification of the target. Thus, hot-start PCR can improve the yield of specific PCR products.
The most common hot-start approaches involve the reversible inactivation or physical separation of one or more critical components in the reaction. The DNA polymerase can be kept in an inactive state at lower temperature by binding to an oligonucleotide, also known as an aptamer, or by binding to an antibody. This bond is disrupted at higher temperatures, releasing the functional polymerase. The polymerase also can be maintained in an inactive state through chemical modification.
Amplification of long DNA fragments is described for numerous applications such as physical mapping applications and direct cloning from genomes. While basic PCR works well when smaller fragments are amplified, amplification efficiency (and therefore the yield of amplified fragments) decreases significantly as the amplicon size increases over 5kb. This decrease in yield can be attributed to the accumulation of truncated products, which are not suitable substrate for additional cycles of amplification. These products often appear as smeared bands on a gel.
PCR of longer products can be achieved using a mixture of two thermostable polymerases. The first polymerase lacks a 3’ – 5’ exonuclease (proofreading) activity; the second enzyme, present at a reduced concentration, contains a potent proofreading activity. Presumably, when the nonproofreading DNA polymerase (e.g., Taq DNA polymerase) misincorporates a dNTP, subsequent extension of the newly synthesized DNA either proceeds slowly or stops completely. The proofreading polymerase (e.g., pfu DNA polymerase) removes the misincorporated nucleotide, allowing the DNA polymerases to continue extension of the new strand.
Other conditions that can also have a significant impact on the yield of longer PCR products are as follows:
- Longer extension times can increase the yield of longer PCR products because fewer partial products are synthesized. Extension times depend on the length of the target; times of 10-20 minutes are common.
- Template quality is crucial. Depurination of the template, which occurs at elevated temperatures and lower pH, will result in more partial products and decreased overall yield. In long PCR, denaturation time is reduced to 2-10 seconds to decrease depurination of the template.
- Additives, such as glycerol and dimethyl sulphoxide (DMSO), also helps lower the strand-separation and primer-annealing temperatures, alleviating some of the depurination effects of high temperatures.
- Reducing potassium concentrations in the reaction buffer by 10-40% can increase the amplification efficiency of longer products.