polymerase chain reaction, many copies of regions of the genome for a number of
ID: 172591 • Letter: P
Question
polymerase chain reaction, many copies of regions of the genome for a number of different downstream applications. In this Project you are PCR-amplifying your mitochondrial HVR1 region so that you can obtain the DNA sequence for that region. The ultimate goal is to determine your haplotype or haplogroup based on that region, which will provide you with insight into your deep ancestry. consider the class discussions you had on PcR this week. Use this information to answer the following questions: 1. What are the primary components of a typical PcR reaction, and what does each of these components do? 2. How can these components be adjusted to optimize, or troubleshoot a reaction that may not be yielding positive results? 3. What are the main steps of a thermal cycling profile, and how can these steps be adjusted to optimize, or troubleshoot, a reaction that may not be yielding positive results? Page 1 of 8Explanation / Answer
1.Main PCR componets:
thermostable DNA polymerase- It is the main polymerizing enzyme use for PCR.Example-Taq DNA polymerase, PFu polymerase. All the dna polymerases used in the PCR reaction must be thermostable because the PCR reaction starts with the denaturation (95oC) of DsDNA molecule.
a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
dNTP- deoxy nucleotide triphosphate is the mixture of all nucleotide triphosphates( dATP,dCTP,dTTP and dGTP) to provide the nucleotide for the DNA synthesis.
DNA primer. Short complemetry sequence of the DNA to start the reaction
Template DNA- is the DNA whose amplification is to be done.
Thermocycler (PCR) machine- to run the reaction setup
PCR tubes- where the reaction mixture to be prepared
2. ALl the componets of PCR like polymerase, dNTP, primer, and template should be added in a appropiate ratio. usually the concentration of template DNA should be more than 1ng, PCR primers should be 25uM, dNTP0.5mM and rest enzyme, its buffer and water.
3.the main steps of a PCR reaction is denaturation of DNA at 95oC followed by annealing of primers to the seperated DNA starnds at 50oC-65oC (annealing is also depends upon the CG content of the Primer.
Extension/elongation: The optimum activity temperature for Taq polymerase is approximately 75–80 °C (167–176 °F), though a temperature of 72 °C (162 °F) is commonly used with this enzyme. In this step, the DNA polymerase synthesizing a DNA strand complementary to the DNA template used in the reaction by adding free dNTPs from the reaction mixture.The processes of denaturation, annealing and elongation constitute a single cycle
for the optimization of PCR one should optimise for the annealing temperature and the extention temperature and time.
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