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  • Polymerase chain reaction (PCR) is a common and essential technique in molecular biology, which can amplify a single or a few copies of a DNA fragment by several orders of magnitude to generate thousands to millions of copies of a specific DNA sequence. PCR rapidly becomes one of the most widely used techniques in laboratories because of its speed, cheapness, and simplicity. PCR raises the analysis of trace amounts of genetic material to a new level of precision and reliability. This rapid, easy method for generating unlimited copies of any fragment of DNA can amplify specific DNA fragments from a small amount of source DNA material, even if the quality of source DNA is relatively poor.

    PCR is a chain reaction in which a single DNA molecule is used to produce two copies, then four, then eight, and so on. This continuous doubling process is accomplished by polymerases, which are able to string together individual DNA building blocks to form long molecular strands. The function of the polymerase requires the building blocks of DNA, such as nucleotides composed of the four bases adenine (A), thymine (T), cytosine (C), and guanine (G). The reaction also requires a small fragment of DNA, called primers, to which they attach these building blocks as well as a longer DNA molecule to serve as a template for constructing a new strand. If these three ingredients are supplied, the polymerases will construct exact copies of the templates. PCR products can be digested with restriction enzymes, sequenced, or cloned.

    The PCR technique involves three main steps: denaturation, annealing, and extension. To amplify a segment of DNA by PCR, the sample is first heated. At high temperatures, the DNA degenerates and separates into two single-stranded DNA. In the annealing step, the reaction is cooled to 50-65 °C, which enables the primers to attach to a specific location on the single-stranded template DNA through hydrogen bonding. In the third step, the end of the annealing primer is extended to form a complementary strand of DNA copies. Polymerase adds the available nucleotides to the ends of the annealed primers to synthesize two new strands of DNA, using the original strand as a template. This process results in replication of the original DNA, with each new molecule containing an old and a new strand of DNA. Each strand can then be used to create two new copies.

    In molecular biology, real-time polymerase chain reaction (also known as quantitative PCR, qPCR) is a technology based on the PCR for the amplification and quantification of target DNA molecules. The advantage of real-time PCR over conventional PCR is that it allows the product to be analyzed while the reaction is in progress. This is achieved by using various fluorescent dyes that react with amplification products and can be measured by instruments. Real-time PCR is the method of choice for quantitative determination of the starting amount of DNA, cDNA, or RNA. Reverse transcriptase polymerase chain reaction (RT-PCR) is a modified PCR technique that uses RNA as the template for in vitro nucleic acid amplification. Reverse transcriptase is an RNA-dependent DNA polymerase and plays a key role in RT-PCR. The enzyme catalyzes DNA synthesis using RNA as the template to generate complementary DNA (cDNA). Because cDNA is not subject to RNase degradation, it is more stable than RNA and can serve as a template for exponential amplification using PCR.

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