Running head: Title of Paper 1

Title of the Paper

Student Name

Institutional Affiliation
Title of Paper 2

a) Primer Placement for Amplification:

Primers should be placed at the ends of the 500 bp region to be amplified. One primer

should bind at the start of the shaded region on one strand, and the other at the end of the

shaded region on the complementary strand, both facing inwards. The vertical ticks indicate

base pairing to the specific sequences within the 500 bp region.

b) Components Required for PCR:

 DNA template: The DNA sequence to be amplified.

 Primers: Short DNA oligonucleotides that flank the region of interest.

 DNA polymerase: An enzyme that synthesizes new DNA strands.

 Nucleotides (dNTPs): The building blocks of DNA (dATP, dCTP, dGTP, dTTP).

 Buffer: Provides the optimal conditions for the activity of DNA polymerase.

 MgCl2: A cofactor required for the activity of many DNA polymerases.

c) Time for Extension Step in PCR:

 With a processivity of 10 bp, DNA polymerase adds nucleotides at a rate of 1 ms per

nucleotide.

 For a 500 bp region, it would take 500 ms or 0.5 seconds to complete synthesis.

 Therefore, 15 seconds is more than sufficient for the extension step.

 Even with a processivity of 100 bp, 15 seconds is still sufficient.

d) Requirement of Topoisomerase in PCR:

PCR does not require topoisomerase. Topoisomerases are enzymes that alter the

supercoiled form of DNA. PCR involves the amplification of linear DNA segments and does

not involve the unwinding of supercoiled DNA, thus making topoisomerase unnecessary.
Title of Paper 3

e) DNA Strands After the First Cycle:

 The first cycle results in two new strands: one that extends from the forward primer to

the end of the template and another from the reverse primer to the other end of the

template.

f) DNA Strands After the Second Cycle:

In the second cycle, these strands serve as templates, resulting in four strands:

1. Two strands of the same length as the original DNA template, each starting and

ending at the respective primers.

2. Two strands of the exact length of the target region (500 bp), each starting from one

primer and ending at the other.

The key feature of the second cycle is the production of strands that are exactly the

length of the region between the two primers. These strands will become the predominant

product in subsequent cycles.

For a clearer understanding, let's create a specific diagram showing the DNA strands

present at the end of the second PCR cycle, with the primer locations indicated.
Title of Paper 4

The diagram above illustrates the DNA strands present at the end of the second cycle

of the Polymerase Chain Reaction (PCR). It shows the four DNA strands that result from this

cycle: two strands are of the same length as the original DNA template, each starting and

ending at the respective primers, and two strands are of the exact length of the target region

(500 bp), each starting from one primer and ending at the other. The locations of the forward

and reverse primers on these strands are also indicated. This visual helps in understanding the

progression of PCR and the amplification of the target DNA region.

g) Majority DNA Products After 20 Cycles:

After 20 cycles, the majority of the DNA products would be fragments of the 500 bp target

region. These would be double-stranded DNA fragments flanked by the primer sequences.

h) Impact of Using ddGTP in Place of dGTP:

Using 2’,3’-dideoxyguanosine 5’-triphosphate (ddGTP) would result in chain

termination. ddGTP lacks a 3' OH group necessary for forming a phosphodiester bond with

the next nucleotide, thus terminating DNA synthesis. After 20 cycles, there would be various

lengths of incomplete DNA strands, none of which would be the full 500 bp target.
Title of Paper 5

i) Adjusting Reaction Parameters for Mismatched Primers:

1. Increase the Annealing Temperature: A higher annealing temperature can increase

the specificity of primer binding, compensating for mismatches.

2. Decrease the Extension Time: Shortening the extension time can prevent non-

specific binding of mismatched primers.

j) Consequences of Using Faulty Primers in Diagnostic Test:

Using faulty primers, even with adjusted conditions, may result in non-specific amplification

or incomplete amplification of the target region. This can lead to false negatives or false

positives in the diagnostic test, compromising its reliability and accuracy.