Polymerase chain reaction ( PCR)

Polymerase chain reaction ( PCR), a technique used to make numerous copies of a specific segment of DNA quickly and accurately. The polymerase chain reaction enables investigators to obtain the large quantities of DNA that are required for various experiments and procedures in molecular biology, forensic analysis, evolutionary biology, and medical diagnostics.

PCR was developed in 1983 by Kary B. Mullis, an American biochemist who won the Nobel Prize for Chemistry in 1993 for his invention. Before the development of PCR, the methods used to amplify, or generate copies of, recombinant DNA fragments were time-consuming and labour-intensive. 

PRINCIPLE

PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp) 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 become limiting as the reaction progresses.

The PCR technique is based on the natural processes a cell uses to replicate a new DNA strand. Only a few biological ingredients are needed for PCR.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
• two DNA primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strands of the DNA target (DNA polymerase can only bind to and elongate from a double-stranded region of DNA; without primers there is no double-stranded initiation site at which the polymerase can bind); 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 (sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), 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 (Mg) or manganese (Mn) 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 (K) ions

The reaction is commonly carried out in a volume of 10–200 μL in small reaction tubes (0.2–0.5 mL volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. 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

PCR is a three-step process that is carried out in repeated cycles. The initial step is the denaturation, or separation, of the two strands of the DNA molecule. This is accomplished by heating the starting material to temperatures of about 95 °C (203 °F). Each strand is a template on which a new strand is built. In the second step the temperature is reduced to about 55 °C (131 °F) so that the primers can anneal to the template. In the third step the temperature is raised to about 72 °C (162 °F), and the DNA polymerase begins adding nucleotides onto the ends of the annealed primers. At the end of the cycle, which lasts about five minutes, the temperature is raised and the process begins again. The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount of DNA.

In the original PCR procedure, one problem was that the DNA polymerase had to be replenished after every cycle because it is not stable at the high temperatures needed for denaturation. This problem was solved in 1987 with the discovery of a heat-stable DNA polymerase called Taq, an enzyme isolated from the thermophilic bacterium Thermus aquaticus, which inhabits hot springs. Taq polymerase also led to the invention of the PCR machine.Because DNA from a wide range of sources can be amplified, the technique has been applied to many fields.

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 (see figure below). The cycling is often preceded by a single temperature step at a very high temperature (>90 °C (194 °F)), 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 (T_m) of the primers.The individual steps common to most PCR methods are as follows:

• 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 94–96 °C (201–205 °F), or 98 °C (208 °F) 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 94–98 °C (201–208 °F) 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 50–65 °C (122–149 °F) 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.

It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. If it is too high, the primer may not bind at all. A typical annealing temperature is about 3–5 °C below the T_m of the primers used. Stable hydrogen bonds between complementary bases are formed only when the primer sequence very closely matches the template sequence. During this step, the polymerase binds to the primer-template hybrid and begins DNA formation.

• Extension/elongation: The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for the thermostable DNA polymerase of Taq (Thermus aquaticus) polymerase is approximately 75–80 °C (167–176 °F),though a temperature of 72 °C (162 °F) 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 are 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 (elongating) 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 (i.e., if there are no limitations due to limiting substrates or reagents), 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 (geometric) amplification of the specific DNA target region.

The processes of denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required to amplify the DNA target to millions of copies. The formula used to calculate the number of DNA copies formed after a given number of cycles is 2ⁿ, where n is the number of cycles. Thus, a reaction set for 30 cycles results in 2³⁰, or 1,073,741,824, copies of the original double-stranded DNA target region.

• Final elongation: This single step is optional, but is performed at a temperature of 70–74 °C (158–165 °F) (the temperature range required for optimal activity of most polymerases used in PCR) 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 4–15 °C (39–59 °F) for an indefinite time, and may be employed for short-term storage of the PCR product

To check whether the PCR successfully generated the anticipated DNA target region (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis may be employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder, a molecular weight marker which contains DNA fragments of known size run on the gel alongside the PCR products.

Stage

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 (assuming 100% reaction efficiency). After 30 cycles, a single copy of DNA can be increased up to 1,000,000,000 (one billion) copies. In a sense, then, the replication of a discrete strand of DNA is being manipulated in a tube under controlled conditions.^[15] 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.