Polymerase Chain Reaction (PCR) + Gel Electrophoresis Flashcards
To know the steps in PCR To be able to explain the steps in PCR Know tools used in PCR Know purpose of tools
What are the steps in PCR?
Denaturation - The two DNA strands are separated
Annealing - Primers are hybridized/joined to the DNA template
Extension - Addition of correct complementary nucleotides into the new strands of DNA through the use of DNA polymerase
Explain the steps in PCR.
Polymerase Chain Reaction consists of 3 steps, Denaturation, Annealing, and Extension.
Firstly, Denaturation is the process of separating the two complementary strands of the DNA. template. This is achieved by heating up the DNA template to break the hydrogen bonds that hold the complimentary strands together.
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Next, annealing comprises of the hybridization of primers to the DNA template. Primers attach to specific sites on the DNA that is to be amplified. During annealing, the temperature of the DNA template is brought down to enable hydrogen bonds to form between the primers and the DNA template, joining them both together.
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Lastly, extension involves the synthesis of new copies of DNA through the use of thermostable DNA Polymerase. Taq polymerase adds complementary nucleotides to the separated DNA strands, resulting in new copies of DNA.
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This cycle is then repeated 30 times. Each cycle uses DNA copies from the previous cycle, leading to an exponential increase in DNA copies
What tools are needs for PCR?
DNA Template
Primers
Thermostable Polymerase
Excess dNTPs (Excess nucleotides)
Buffer
Why is Thermostable Polymerase used? (Taq Polymerase)
Thermostable DNA Polymerase is used as it will not denature during the temperature changes of PCR cycles due to its ability to withstand high temperatures.
What is the function of primers?
Primers bind to specific sites of the DNA to be amplified and are complimentary to the opposite ends of the target DNA sequence.
Why do DNA molecules migrate from negative to positive during Gel Electrophoresis?
DNA molecules are negatively charged. Since opposite charges attract each other, DNA molecules migrate from negative to positive.
