Course: SAT 170 Introductory Biotechnology

Unit 6 Reading and Multimedia Assignment

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“Polymerase chain reaction (PCR) ” by Boundless, CC BY SA 3.0

https://www.boundless.com/biology/biotechnology/dna-and-biotechnology/polymerase-chain-reaction-pcr/

Reading Assignment: PCR
Polymerase chain reaction (PCR) is a technique that amplifies a selected target in a DNA molecule very rapidly.

PCR Reaction

The polymerase chain reaction is a technique for quickly "cloning" a particular piece of DNA in the test tube (rather than in living cells like E. coli). Thanks to this procedure, one can make virtually unlimited copies of a single DNA molecule in a matter of hours, even though it is initially present in a mixture containing many different DNA molecules. Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.

The method relies on thermal cycling; cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. Regular DNA polymerase cannot be used because it would be degraded by the high temperatures necessary for the reaction to proceed.

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a 40,000 year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar.

In order to perform PCR, at least a portion of the sequence of the DNA molecule that is to be replicated must already be known, in order to synthesize primers. Primers are short oligonucleotides (containing about two dozen nucleotides) that are precisely complementary to the sequence at the 3' end of each strand of the DNA you wish to amplify, so they are extremely specific. In order to ensure specificity, they should also be at least 15 base pairs long. The DNA template is heated to separate its strands and mixed with the primers. If the primers find their complementary sequences in the DNA, they bind to them, a step known as annealing.

Synthesis begins, as always 5' -> 3', using the original strand as the template, a step known as extension. The reaction mixture must contain all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) a DNA polymerase. Polymerization continues until each newly-synthesized strand has proceeded far enough to contain the site recognized by the other primer, creating two DNA molecules identical to the original molecule. These two molecules are then heated to separate their strands, and the process is repeated (see above figure).

After the third cycle, 25% of the molecules produced will be identical to the target sequence. From this point onward, each subsequent cycle doubles the number of DNA molecules. The number of DNA molecules produced can therefore be written as 2n, where n equals the number of cycles in the reaction. Therefore, one billion molecules can be produced after 30 cycles.

PCR is not error-free. The polymerase can make substitutions in the nucleotides as it synthesizes each strand, so that the final population of PCR products may be slightly different than the original template. For this reason, gene cloning should still be used when large amounts of a gene are needed, to ensure that some correct copies are obtained. It is therefore also important to check the accuracy of the insert sequence when cloning from a PCR product.