polymerase Chain Reaction

It is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on the use of the ability of DNA polymerase to synthesize a new DNA strand that is complementary to the offered template strand. Since DNA polymerase can only add one nucleotide to an already existing 3`OH group, it needs a primer to which it can add the first nucleotide. This requirement allows the delineation of a specific region of the template sequence that the investigator wishes to amplify. At the end of the PCR reaction, the specific sequence accumulates in billions of copies (amplicons).

Components of PCR

DNA template: the DNA sample that contains the target sequence. At the beginning of the reaction, the original double-stranded DNA molecule is exposed to a high temperature to separate the strands from each other. DNA polymerase is a type of enzyme that synthesizes new DNA strands that are complementary to the target sequence. The first and most widely used of these enzymes is TaqDNA polymerase (from Thermis aquaticus), while PfuDNA polymerase (from Pyrococcus furiosus) is widely used due to its greater precision in DNA copying. Although these enzymes differ slightly, they both have two capabilities that make them suitable for PCR: 1) they can generate new DNA strands using a DNA template and primers, and 2) they are heat resistant, priming short single-stranded pieces. DNAs that are complementary to the target sequence. The polymerase begins at the end of the primer with the synthesis of new DNA. Nucleotides (dNTPs or deoxynucleotide triphosphates) individual units of the bases A, T, G and C, which are essentially “building blocks” for new DNA strands. Reverse transcription PCR) is a PCR that converts the RNA sample into cDNA using the enzyme.

Limitations of PCR and RTPCR The PCR reaction begins to make exponential copies of the target sequence. Back extrapolation to the initial amount of the target sequence contained in the sample is only possible during the exponential phase of the PCR reaction. Over time, due to inhibitors of the polymerase reaction found in the sample, limitation of the reagent, accumulation of pyrophosphate molecules, and self-adhesion of the accumulated product, the PCR reaction stops amplifying the target sequence to an exponential speed and a “plateau” occurs. Effect “on, making end point quantification of PCR products unreliable. This is the attribute of PCR that makes quantitative real-time RTPCR so necessary.

Application

PCR allows the isolation of DNA fragments from genomic DNA by selective amplification of a specific DNA region. This use of PCR expands many pathways, such as the generation of hybridization probes for Southern or Northern hybridization and DNA cloning, that require larger amounts of DNA representing a specific region of DNA. PCR provides these techniques with large amounts of pure DNA and allows DNA samples to be analyzed even from very small amounts of starting material. Other uses of PCR include DNA sequencing to determine unknown PCR amplified sequences in which one of the amplification primers can be used in Sanger sequencing, isolation of a DNA sequence to accelerate recombinant DNA technologies that allow the insertion of a DNA sequence into a plasmid, phage or cosmid (depending on size) or the genetic material of another organism. Bacterial colonies (such as E. coli) can be quickly screened for correct DNA vector constructs by PCR. [20] PCR can also be used for genetic fingerprinting; forensic technique used to identify a person or organism by comparing experimental DNA using various PCR-based methods. Electrophoresis of PCR amplified DNA fragments: Father Child Mother The child has inherited some, but not all, of the fingerprints of each of her parents, giving them a new and unique fingerprint. Some PCR fingerprinting methods are highly discriminatory and can be used to identify genetic relationships between individuals, such as parents and children or between siblings, and are used in paternity

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