Question

Please answer both questions:

1. A number of different buffers/components are used in the PCR. Explain the function of each:

DNA plasmid

Primer pair

dNTPs mixture

Taq polymerase

2. In our PCR reaction the optimum annealing temperature is 56^oC. Explain the general factors that help determine the optimum annealing temperature of a PCR reaction

Answer 1

1.DNA plasmid-

-A target gene inserted into a circular piece of DNA called a plasmid.

-plasmid is introduced into bacteria by a process called transformation, and bacteria carrying the plasmid are selected using antibiotics.

-Bacteria with the correct plasmid are used to make more plasmid DNA induced to express the gene and make protein

PRIMER PAIR-

Primers are the strands of DNA (or RNA) that serve as this ini for the DNA replication process, and they are used to demarcate the segment of the DNA template to be amplified.

In the PCR process, two primers are matched to the segment of DNA. During the annealing process, one primer attaches to the top strand and the other attaches to the bottom strand at each end of the sample. primers composed of RNA can be used, DNA primers are more commonly seen in PCR as they are more temperature-stable, which is important during the annealing process.

Primer Pairs

Primer pairs consist of the upper and lower primers. Although two different primers may make up the primer pairs, the primers both have similar melting temperatures in order to facilitate a successful PCR reaction. Software is available which identifies potential primer pairs, providing their melting and annealing temperatures.

dNTPs MIXTURE-

dNTPs in PCR allow the extension of primed DNA strand.once the double stranded has been separated and the primers are annealed the polymerase( usuallyTAC1) will ise these dNTPs to extend a strand of DNA that is complimentary to the rimed strand of DNA.

The name "de-oxy " refers to the missing oxygen of hydroxyl group on the3' end of the sugar structure(ribose).

Taq POLYMERASE-

PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, after heat-tolerant bacterium from which it was isolated (Thermus aquaticus).

2.OPTIMUM ANNEALING TEMPERATURE OF A PCR REACTION-

• Primer Tm and annealing temperatures (Ta) values should be determined using PrimerDigital’s Tools;
• Non-specific product formation can often be avoided by optimizing the annealing temperature or by switching to a hot start enzyme;
• Ta can be optimized by doing a temperature gradient PCR, starting at 5°C below the lowest Tm of the primer pair;
• Ideally, primer Tm values should be near to the extension temperature. If Tm values are calculated to be greater than the extension temperature, a two-step PCR program (combining annealing and extension into one step) can be employed;
• The most important values for estimating the Ta is Tm and CG% of the primers and the length of PCR fragment (L);
• Primers with high Tm's (> 60°C) can be used in PCRs with a wide Ta range compared to primers with low Tm's (< 50°C);
• The optimal annealing temperature for PCR is calculated directly as the value for the primer with the lowest Tm (T_m^min):
• 
  
• where L is length of PCR fragment.