Nucleic Acid Fragment Amplification by Polymerase Chain Reaction

Illustration shows the amplification of a D N A sequence by the polymerase chain reaction. P C R consists of three steps; denaturation, annealing, and D N A synthesis; that occur at high, low, and intermediate temperatures. In step 1, the denaturation step, the sample is heated to a high temperature so the D N A strands separate. In step 2, annealing, the sample is cooled so two primers can anneal to the two strands of D N A. The primers are spaced such that the sequence of interest between them will be amplified. In step 3, D N A synthesis, the sample is warmed to the optimal temperature for Taq polymerase, which synthesizes the complementary strand from the primer to the 3 prime end of the molecule. This cycle is repeated again and again. Each time, the newly synthesized strands serve as templates so that the amount of D N A doubles with each cycle. As the cycles continue, more and more strands are the size of the distance between the two primers; in the end, the vast majority of strands are this size.
Scientists use polymerase chain reaction, or PCR, to amplify a specific DNA sequence. Primers—short pieces of DNA complementary to each end of the target sequence combine with genomic DNA, Taq polymerase, and deoxynucleotides. Taq polymerase is a DNA polymerase isolated from the thermostable bacterium Thermus aquaticus that is able to withstand the high temperatures that scientists use in PCR. Thermus aquaticus grows in the Lower Geyser Basin of Yellowstone National Park. Reverse transcriptase PCR (RT-PCR) is similar to PCR, but cDNA is made from an RNA template before PCR begins. Source: OpenStax Biology 2e

OpenStax Biology 2e

Although genomic DNA is visible to the naked eye when it is extracted in bulk, DNA analysis often requires focusing on one or more specific genome regions. Polymerase chain reaction (PCR) is a technique that scientists use to amplify specific DNA regions for further analysis. Researchers use PCR for many purposes in laboratories, such as cloning gene fragments to analyze genetic diseases, identifying contaminant foreign DNA in a sample, and amplifying DNA for sequencing. More practical applications include determining paternity and detecting genetic diseases.

DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR). The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.

Source:

Clark, M., Douglas, M., Choi, J. Biology 2e. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/biology-2e


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