Polymerase Chain Reaction (PCR ) is a technique that utilizes the enzyme DNA polymerase for making copies of a gene, gene fragment, or specific DNA or RNA sequence. DNA polymerase is the enzyme that catalyzes replication and repair of DNA. PCR is a requisite tool in research, clinical, environmental, and forensic laboratories. PCR employs cycles of heating and cooling to first melt and separate the double-stranded DNA containing the sequence of interest, then, with the help of specially designed sequences called primers, locate the target sequence, and exponentially produce new copies. PCR is a versatile and adaptable technique, with many variations and applications. Since its introduction in the mid eighties, PCR has modernized the field of molecular biology. Procedures that once took weeks to produce results can now be accomplished in a few hours.
PCR is often used to amplify an unknown sequence. However, in order to construct complementary primers, some information must be known about sequences bordering the region of interest. Primers are annealed to the sequences that flank the sequence to be amplified, in order to initiate replication of the target DNA. The most commonly used DNA polymerase for PCR is Taq polymerase, isolated from the bacterium Thermus aquaticus, known for its supreme stability at high temperatures. Deoxyribonucleotide triphosphates (dNTPs), the basic units of DNA, are another key component in the PCR mixture. In a small tube the template DNA, water, dNTPs, DNA polymerase, primers, and a salt such as magnesium chloride are briefly centrifuged, along with a control tube with template DNA omitted, and placed in a PCR thermocycler.
A typical PCR run includes a series of 20 to 40 cycles of heating and cooling, separating, then annealing. The first denaturation step brings the reaction mixture to a temperature between 94 and 98C, for 20 to 30 seconds. The heat disrupts the hydrogen bonds between complementary bases on the DNA strand, resulting in two single strands. The second step is the primer annealing step, where the reaction temperature is decreased to between 50 and 65C, for 20 to 40 seconds, to allow annealing of primers to the single-stranded DNA, resulting in a DNA-primer hybrid. Polymerase then binds to the DNA-primer hybrid, and DNA synthesis commences. The amount of target DNA doubles with each subsequent cycle, resulting in exponential amplification of the target DNA.
PCR has many applications. In clinical settings PCR can be applied for rapid diagnosis of infectious disease, or to detect mutations associated with diseases such as cancer. The procedure can be utilized to ferret out bacteria that are difficult to culture, and is often more specific and accurate than standard diagnostic tests because it can zero in on the distinctive DNA of a particular infectious organism. In forensic laboratories DNA from the tiniest of samples, a skin fragment, a single hair, or a drop of blood, can generate a genetic fingerprint. In paternity and genetic testing PCR is used to examine repeat base pair sequences on DNA segments. PCR is used in the study of evolution and in historical medical genetics. Other applications include cloning of DNA for sequencing, and the study of evolutionary relationships between organisms.
There are several types of PCR, with new variations always on the horizon. Allele-specific PCR is used to identify or recruit single-nucleotide polymorphisms (SNPs), variations in a single nucleotide between members of a species, and is especially useful in genotyping applications. Reverse Transcription PCR (RT-PCR) can isolate or identify a known sequence from RNA using RNA-directed polymerase (reverse transcriptase), an enzyme found in retroviruses. RNA is first transcribed into a complementary strand of DNA (cDNA), followed by a standard PCR procedure. Quantitative real-time PCR (QRT-PCR) has many applications. It uses fluorescent dyes containing DNA probes to determine the presence of a DNA sequence and to quantify the amplified product. Assembly PCR is used to synthesize a long DNA sequence from smaller DNA segments, and is valuable in research laboratories for protein expression studies. Asymmetric PCR provides preferential amplification of one strand of DNA over another, utilizing a large excess of primers to produce the favored strand. More cycles are required for asymmetric PCR because the amplification process takes longer than a traditional PCR run. Nested PCR can be applied when greater specificity is desired, using two sets of primers, and two PCR reactions. The first reaction, using one pair of primers, generates the DNA product. A second PCR run employs primers with different binding sites to generate a specific DNA product. Helicase-dependent PCR is a variation that utilizes DNA helicase, the unwinding enzyme, instead of heat denaturation, and a constant temperature instead of heating and cooling cycles. In hot-start PCR the reaction components are heated prior to addition of polymerase, to reduce non-specific amplification. Intersequence-specific (ISSR) PCR amplifies regions between specific sequence repeats, and is used in DNA fingerprinting. Inverse PCR is an option when only one flanking sequence is known. Ligation-mediated PCR links small pieces of DNA to the target sequence using multiple primers. This method can be applied in genome walking, to identify unknown flanking regions of a known sequence, and in DNA footprinting, to study DNA-protein interactions. Multiplex-PCR uses multiple primers to produce DNA of varying lengths and can target multiple genes at once. It is useful in research and in rapid diagnosis, and can yield a large amount of information in a single test, saving time and resources.
PCR provides for easy and inexpensive DNA copying, allowing unlimited reproduction of genetic material. PCR requires a few key components, test tubes, a centrifuge, and a thermocycler, and can produce millions of copies of a small DNA fragment, all in a few hours time. After twenty plus years, new applications and modifications continue to be adapted and perfected. Molecular biologists who routinely employ PCR continue to seek new and improved methods to circumvent specific challenges, such as the need for faster reactions, greater primer specificity, a reduced reliance on thermocyclers, less contamination, and a greater ability to work with DNA samples that are damaged or from an unknown source. New technologies to replace PCR are in development; however, most are still beleaguered by daunting technological hurdles. PCR is not going anywhere anytime soon, and will likely remain the gold standard in molecular biology for the foreseeable future.
