polymerase chain reaction

polymerase chain reaction
Name	polymerase chain reaction
Caption	A modern thermal cycler used to automate the temperature cycles required.
Acronym	PCR
Inventor	Kary Mullis
Manufacturer	Various, including Cetus Corporation
Related	RT-PCR, qPCR, dPCR

polymerase chain reaction is a fundamental technique in molecular biology that amplifies a single or few copies of a specific segment of DNA across several orders of magnitude. Developed in the 1980s, it revolutionized fields from genetic research to forensic science and clinical diagnostics. The method relies on thermal cycling, involving repeated heating and cooling for DNA melting and enzymatic replication, using a thermostable DNA polymerase.

Principle and mechanism

The core principle is the exponential amplification of a DNA target sequence through repeated cycles of three temperature-dependent steps. The process begins with denaturation, where the double-stranded DNA is heated to separate it into two single strands. During annealing, the temperature is lowered to allow short DNA primers to bind to complementary sequences flanking the target region. Finally, in the extension step, a thermostable DNA polymerase, such as Taq polymerase from Thermus aquaticus, synthesizes a new DNA strand by adding nucleotides. Each cycle theoretically doubles the amount of the target DNA fragment, leading to millions of copies after 20-40 cycles.

History and development

The concept was conceived by Kary Mullis in 1983 while he was working for the Cetus Corporation in Emeryville, California. Mullis's key insight was using two opposing DNA primers and a heat-stable enzyme to enable automated cycling. The first publication demonstrating a functional method appeared in 1985 in the journal Science. The subsequent adoption of Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus by researchers like Alice Chien, greatly improved efficiency and specificity. For this revolutionary invention, Kary Mullis was awarded the Nobel Prize in Chemistry in 1993.

Components and reagents

A standard reaction mixture requires several key components. The DNA template contains the target sequence to be amplified. Two synthetic oligonucleotide primers, typically 18-25 bases long, define the boundaries of the amplification. The enzyme DNA polymerase, most commonly Taq polymerase, catalyzes the synthesis of new DNA strands. Deoxynucleotide triphosphates (dNTPs) serve as the building blocks for the new DNA. The reaction occurs in a buffer solution that provides optimal pH and chemical conditions, often containing magnesium chloride as a cofactor for the polymerase. The mixture is placed in a specialized instrument called a thermal cycler.

Procedure and steps

The procedure is highly automated within a thermal cycler. An initial step, often called hot-start PCR, may be used to prevent non-specific amplification by activating the polymerase only at high temperature. The core cycling then begins, typically repeating 25-40 times. Each cycle consists of the denaturation step at 94–98°C, annealing at 50–65°C (temperature specific to the primer sequences), and extension at 72°C. Following the cycles, a final extension step ensures all amplicons are fully synthesized. The products, or amplicons, are then analyzed using techniques like agarose gel electrophoresis or DNA sequencing.

Variations and applications

Numerous specialized variants have been developed to extend its utility. RT-PCR first uses reverse transcriptase to convert RNA into complementary DNA (cDNA) for amplification, crucial for studying gene expression. Real-time PCR or qPCR allows for the quantification of the DNA target during the reaction using fluorescent dyes. Digital PCR partitions the sample for absolute quantification. Applications are vast, including genetic fingerprinting in forensic science, prenatal diagnosis of genetic disorders, detection of pathogens like SARS-CoV-2, archaeogenetics, and basic research in genomics.

Limitations and considerations

Despite its power, the technique has several limitations. It is highly sensitive to contamination from extraneous DNA, requiring stringent laboratory practices. The fidelity of the DNA polymerase can introduce errors, especially with enzymes lacking proofreading activity. The requirement for prior sequence knowledge to design DNA primers prevents its use for discovering entirely unknown sequences. Amplification efficiency can be inhibited by contaminants in the sample, such as heme in blood or humic acid in soil. Furthermore, it amplifies only short DNA fragments, typically up to a few kilobases, limiting its use for larger genomic regions.

Category:Molecular biology techniques Category:Laboratory techniques Category:DNA