Polymerase chain reaction


The polymerase chain reaction is a laboratory method widely used to amplify copies of specific DNA sequences rapidly, to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.
PCR is fundamental to many of the procedures used in genetic testing, research, including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research and forensic science.
The majority of PCR methods rely on thermal cycling. Thermal cycling exposes reagents to repeated cycles of heating and cooling to permit different temperature-dependent reactions—specifically, DNA melting and enzyme-driven DNA replication. PCR employs two main reagents—primers and a thermostable DNA polymerase. In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called nucleic acid denaturation. In the second step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase used was heat-susceptible, it would denature under the high temperatures of the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which was a tedious and costly process.
Applications of the technique include DNA cloning for sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA-based phylogenies, or functional analysis of genes; diagnosis and monitoring of genetic disorders; amplification of ancient DNA; analysis of genetic fingerprints for DNA profiling ; and detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases.

Principles

PCR amplifies a specific region of a DNA strand. Most PCR methods amplify DNA fragments of between 0.1 and 10 kilo-base pairs in length, although some techniques allow for amplification of fragments up to 40 kbp. The amount of amplified product is determined by the available substrates in the reaction, which becomes limiting as the reaction progresses.
A basic PCR set-up requires several components and reagents, including:
  • a DNA template that contains the DNA target region to amplify
  • a DNA polymerase; an enzyme that polymerizes new DNA strands; heat-resistant Taq polymerase is especially common, as it is more likely to remain intact during the high-temperature DNA denaturation process. Pfu polymerase is a heat-resistant polymerase with higher accuracy compared to Taq due to its ability to proofread, but it runs slower. Adding a small amount of Pfu to Taq combines the benefits of both.
  • two DNA primers that are complementary to the 3' ends of each of the sense and anti-sense strands of the DNA target ; specific primers that are complementary to the DNA target region are selected beforehand, and are often custom-made in a laboratory or purchased from commercial biochemical suppliers
  • deoxynucleoside triphosphates, or dNTPs, the building blocks from which the DNA polymerase synthesizes a new DNA strand
  • a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
  • bivalent cations, typically magnesium or manganese ions; Mg2+ is the most common, but Mn2+ can be used for PCR-mediated DNA mutagenesis, as a higher Mn2+ concentration increases the error rate during DNA synthesis; and monovalent cations, typically potassium ions
The reaction is commonly carried out in a volume of 10–200 μL in small reaction tubes in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction. Many modern thermal cyclers make use of a Peltier device, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the device's electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibrium. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermal cyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

Typically, PCR consists of a series of 20–40 repeated temperature changes, called thermal cycles, with each cycle commonly consisting of two or three discrete temperature steps. The cycling is often preceded by a single temperature step at a very high temperature, and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature of the primers. The individual steps common to most PCR methods are : initialization, annealing and extension.
  • Initialization: This step is only required for DNA polymerases that require heat activation by hot-start PCR. It consists of heating the reaction chamber to a temperature of, or if extremely thermostable polymerases are used, which is then held for 1–10 minutes.
  • Denaturation: This step is the first regular cycling event and consists of heating the reaction chamber to for 20–30 seconds. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules.
  • Annealing: In the next step, the reaction temperature is lowered to for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand.
  • Extension/Elongation: The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for the thermostable DNA polymerase of Taq polymerase is approximately, though a temperature of is commonly used with this enzyme. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that is complementary to the template in the 5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy group at the end of the nascent DNA strand. The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify. As a rule of thumb, at their optimal temperature, most DNA polymerases polymerize a thousand bases per minute. Under optimal conditions, at each extension/elongation step, the number of DNA target sequences is doubled. With each successive cycle, the original template strands plus all newly generated strands become template strands for the next round of elongation, leading to exponential amplification of the specific DNA target region.
  • Final elongation: This single step is optional, but is performed at a temperature of for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.
  • Final hold: The final step cools the reaction chamber to for an indefinite time, and may be employed for short-term storage of the PCR products.
Image:Roland Gel.JPG|thumb|Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.
To check whether the PCR successfully generated the anticipated DNA target region, agarose gel electrophoresis may be employed for size separation of the PCR products. The size of the PCR products is determined by comparison with a DNA ladder, a molecular weight marker which contains DNA fragments of known sizes, which runs on the gel alongside the PCR products.

Stages

As with other chemical reactions, the reaction rate and efficiency of PCR are affected by limiting factors. Thus, the entire PCR process can further be divided into three stages based on reaction progress:
  • Exponential amplification: At every cycle, the amount of product is doubled. After 30 cycles, a single copy of DNA can be increased up to 1,000,000,000 copies. In a sense, then, the replication of a discrete strand of DNA is being manipulated in a tube under controlled conditions. The reaction is very sensitive: only minute quantities of DNA must be present.
  • Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents, such as dNTPs and primers, causes them to become more limited.
  • Plateau: No more product accumulates due to exhaustion of reagents and enzyme.