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The polymerase chain reaction (PCR) is a powerful technique developed by Kary Mullis in 1983. It allows for the rapid amplification of a DNA fragment of interest for further study, including many types of genetic analysis.

A PCR reaction typically involves the following components:

• Double-stranded DNA template
• DNA primers complementary to opposite strands and designed to anneal to the template at the ends of the DNA fragment of interest
• Heat-stable DNA polymerase
• dNTPs (a mixture of all four deoxyribonucleotides)
• ${MgCl}_{2}$ ($Mg^{+2}$ is a cofactor required for DNA polymerase activity)

Briefly, PCR begins with denaturation of double-stranded DNA at a temperature between 94-96$^{\circ}$C to make it single-stranded. Then, the temperature is lowered to between 45-68$^{\circ}$C to allow the DNA primers designed to anneal to either end of the gene of interest. The annealing temperature is heavily dependent on the length and GC content of the primers.

Lastly, the temperature is raised to about 72$^{\circ}$C to allow DNA polymerase to elongate from each of the primers, synthesizing the DNA strand complementary to the template. This represents a single cycle of PCR, but this process is repeated between 25-50 more times, with the amount of template doubling during each cycle. The steps involved in each cycle of PCR are shown below.

Suppose you are a laboratory researcher performing PCR to amplify a gene of interest. Following PCR and agarose gel electrophoresis to visualize your PCR product(s), you notice that, while your desired PCR product has been successfully amplified, there are several other bands of incorrect sizes on your gel. While you are generally aware that the concentrations of dNTPs and ${MgCl}_{2}$ affect the fidelity of PCR reactions, you perform an internet search to learn more about their effects more specifically.

Based upon your knowledge of PCR and your own internet search, what are some strategies that you may try to optimize your PCR reaction to increase specificity and eliminate these non-specific PCR products?

Select ALL that apply.

A

Increase the annealing temperature.

B

Decrease the annealing temperature.

C

Increase the elongation temperature.

D

Decrease the denaturation temperature.

E

Increase the $[{MgCl}_{2}]$.

F

Decrease the $[{MgCl}_{2}]$.

G

Reduce the amount of dNTPs.

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