Polymerase chain reaction
PCR consists of three steps:
• Denaturation: Samples of DNA with the target sequence are heated to a reaction temperature of 94-96˚ C that causes DNA melting of the DNA template without denaturing the enzyme by disrupting the hydrogen bonds between complementary bases of the double helical DNA, yielding single-stranded DNA molecules(“How Is PCR (polymerase Chain Reaction) Done?”).
DNA polymerase does not get degraded in such high temperatures since the DNA polymerase used in this reaction is thermostable as it is isolated from thermophilic bacteria, Thermus aquaticus (Taq) in this case( “Process of RDT”)
• Annealing: DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, a primer is required that can add the first nucleotide; this requirement makes it possible to delinate a specific region of template sequence that the researcher wants to amplify(“PCR”). In this step the temperature is lowered to 50-65˚C for one to several minutes which facilitates the primers to pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied) (“How Is PCR (polymerase Chain Reaction) Done?”). On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template (“How Is PCR (polymerase Chain Reaction) Done?”). In a PCR experiment, two primers are designed to match the target template DNA; through complementary base pairing, one primer attaches to the top strand at one end of target template DNA, and the other primer attaches to the bottom strand at the other end (“PCR Virtual Lab”).
• Extension or elongation: The temperature is raised to 72˚C for one to several minutes, this allows Taq polymerase to attach at each priming site and extend (synthesize) a new DNA strand(“Biology Animation Library”). DNA polymerase (naturally-occurring complex of proteins whose function is to copy a cell's DNA before it divides in two) uses the original single strand of DNA as a
• Denaturation: Samples of DNA with the target sequence are heated to a reaction temperature of 94-96˚ C that causes DNA melting of the DNA template without denaturing the enzyme by disrupting the hydrogen bonds between complementary bases of the double helical DNA, yielding single-stranded DNA molecules(“How Is PCR (polymerase Chain Reaction) Done?”).
DNA polymerase does not get degraded in such high temperatures since the DNA polymerase used in this reaction is thermostable as it is isolated from thermophilic bacteria, Thermus aquaticus (Taq) in this case( “Process of RDT”)
• Annealing: DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, a primer is required that can add the first nucleotide; this requirement makes it possible to delinate a specific region of template sequence that the researcher wants to amplify(“PCR”). In this step the temperature is lowered to 50-65˚C for one to several minutes which facilitates the primers to pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied) (“How Is PCR (polymerase Chain Reaction) Done?”). On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template (“How Is PCR (polymerase Chain Reaction) Done?”). In a PCR experiment, two primers are designed to match the target template DNA; through complementary base pairing, one primer attaches to the top strand at one end of target template DNA, and the other primer attaches to the bottom strand at the other end (“PCR Virtual Lab”).
• Extension or elongation: The temperature is raised to 72˚C for one to several minutes, this allows Taq polymerase to attach at each priming site and extend (synthesize) a new DNA strand(“Biology Animation Library”). DNA polymerase (naturally-occurring complex of proteins whose function is to copy a cell's DNA before it divides in two) uses the original single strand of DNA as a