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Molecular Genetic Techniques

Using PCR for DNA Amplification

Polymerase chain reaction (PCR) has many uses in medicine and forensic investigations.

Many advances in DNA technology would have been impossible without DNA amplification, the process by which many copies of a piece of DNA are produced in a laboratory. One successful technique for rapidly reproducing a section of DNA is called polymerase chain reaction (PCR). In this process, DNA is separated into two strands, and each strand is exposed to a DNA primer, which is an anchor and starting point for DNA polymerase to add free nucleotides to a growing strand of DNA, and DNA polymerase, an enzyme that assists in adding free nucleotides to a growing strand of DNA. DNA primers are custom made to match the ends of the regions that are to be amplified. This process allows the DNA strands to replicate using actions similar to those in DNA replication. The primer acts as an anchor to specify the location on a strand that is used as a template for starting to build a new strand of DNA. After one cycle, the original template strand has been copied, resulting in four single strands of DNA. The cycle is repeated, but this time, each of the four single strands of DNA acts as the template strand. With each cycle, the number of copies of DNA grows exponentially (however, it is important to note that the depletion of primers and nucleotides results in amplification of DNA that is more logistic). Billions of copies can be made from a single piece of DNA in a few hours. Once the DNA has been amplified, testing can be run on the copies rather than on the original, reducing the chances of damaging it and allowing for multiple tests to be performed instead of just one.

PCR steps are performed at different temperatures to produce amplified DNA successfully. The three temperature-dependent steps begin with double-stranded DNA being denatured, a process that modifies the DNA structure, which requires a high-temperature environment around 95˚C. The second step, annealing, a process of a primer being paired through hydrogen bonding, usually takes place at a temperature around 55˚C. The final step, extending, which is new DNA synthesis, occurs around 72˚C.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a laboratory technique used to make many copies of a section of DNA. In PCR, many DNA copies can be quickly made by taking advantage of complementary base pairing. At the end of each cycle, each newly formed strand becomes a template strand for the next cycle.
Although PCR is very useful and effective, it cannot replace gene cloning in cells if a large amount of a particular gene is needed. This is because PCR is prone to errors during the rapid replication process and limited to the length of the DNA fragment that can be copied. Instead, PCR is used to provide specific fragments that can then be added to the cloning process. Primers, short DNA strands, are made to include restriction sites. A restriction site is a place on a DNA strand that contains specific sequences of nucleotides and can be recognized by restriction enzymes. The restriction site nucleotide sequences match the cloning locations on each of the cloning vectors. When the primer and the nucleotides are joined, the success rate of producing clones that are free of errors increases.

PCR has shown to be very beneficial in both medical and legal situations. This technique allows a single hair follicle, a drop of blood, or even a single cell to provide enough DNA evidence to test for genetic markers, diagnose genetic disorders, and detect bacteria or viruses.