The polymerase chain reaction, or PCR, allows us to make many copies of targeted

Question

The polymerase chain reaction, or PCR, allows us to make many copies of targeted 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: What are the primary components of a typical PCR reaction, and what does each of these components do? How can these components be adjusted to optimize, or troubleshoot, a reaction that may not be yielding positive results? 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?

Explanation / Answer

1. The primary components of a PCR reaction are:

a. PCR buffer: PCR buffer provides buffereing condition to the system to maintain balance of ions and pH. It also provides MgCl2, which is an essential component of the polymerase enzyme to get activated.

b, dNTPS: dNTPS are the essential component of any PCR reaction as it contains the mixture of ATP, TTP, GTP and CTP. these acts as base during polymerase rection to amplify the target strand.

c. Primers: primers are used for the amplification. It anneals with the template DNA and acts as precursor for the polymerase to act upon it and synthesize the new starnd. Primers are of two types, forward primer and reverse primer. The synthesis of primers are dependent upon the sequence of the DNA template.

d. Taq Polymerase: It the enzyme which helps in synthesis of the newly strand of DNA from the template. It acts from 5' to 3' direction. Usually a thermostable Taq polymearse is used and polymerase 1000 bases/min during PCR reaction.

2. These components can be adjusted to optimize the PCR which may not be yielding positive result are, conc. of MgCl2, Primers concentration, dNTP concentration. An appropriate amount of MgCl2 is required for the enzyme to perform polymerase reaction. without MgCl2 the reaction could be improper leading to negative result. Concentration of primers can also be optimized, the primer concentration should not be too high or too low as that can yield negative result. Usually a concentration of 0.5 mM of each primers are used in any kind of pCR reaction. The amount of dNTP can also effect the PCR reaction. If the dNTP concentration is poor this can lead to no amplification. Usually 1 mM dNTP is used in standard reaction but this can be optimized based on the reaction.

3. Thermal cycle profile have a toatl of five steps: a. Initial denaturation, b. Cycle denaturation, c. Annealing of primers, d. Extension, e. Final extension.

Usually there are 30 cycle erformed during PCR reaction. The steps that can be modified to obtain a positive result are Annealing temperature of the PCR reaction and Extension time of the polymerase. The annealing temperature depends upon the Tm of the forward and reverse primer. It usually 2-4 0C above or below the Tm of both the primers. The extension time will depend upon the length of the template to be amplified, Taq polymerase takes 1 min to synthesize 1000 bases, so the time can be adjusted accordingly.

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