Question

Say you design a PCR experiment with normal temperature settings and reagent amounts (primers, dNTPs, polymerase) for 20 cycles. You want to adjust your experiment so that it will increase the amount of DNA produced. Which of the following adjustments to the experiment will increase the amount of DNA amplified during PCR?

Group of answer choices

You increase the length of the primers.

You increase the temperatures in all of the steps.

None of these adjustments will increase the amount of DNA produced through PCR.

You run 5 extra cycles.

You double the amount of dNTPs.

Answer 1

Answer: option (4) is the correct answer. You run 5 extra cycles and that will increase the amount of DNA amplified during PCR.

Polymerase chain reaction (PCR): PCR is a temperature based technology (also known as thermocycler) which is used to amplify desirable DNA sequences by in vitro replication. PCR tubes containing the DNA mixture of interest with all the required ingredients are kept in the thermocycler machine, and program the machine that changes the temperature to suit each step of the process. PCR is a common tool used in biological and medical research to amplify a desirable gene or DNA sequence in many numbers (thousands to millions) from a very small amount of DNA.

Core ingredients of the PCR are as follows:

1. Template DNA: the DNA template to be copied

2. Primers: short stretches of DNA that initiates the PCR reaction (with the help of DNA polymerase), complementary to the sequence to be amplified. Two DNA primers that are complementary to the 3' end of each of the sense and anti-sense strands of the DNA target. Primers are single strands of DNA sequences with around 20 to 30 bases in length.

3. DNA nucleotide bases: DNA bases (A, C, G and T) are the building blocks of DNA and are needed to construct the new strand of DNA during amplification.

4. Taq polymerase enzyme: key enzyme that carryout the amplification reaction by adding in the new DNA bases.

5. Buffer: it ensures the right conditions for the reaction.

Amplification is carried out in cycles and involves a process of heating and cooling called thermal cycling which is carried out by thermocycler machine. It involves three main steps:

1. Denaturation: in this the DNA sample is heated up to separate the double strands into two single strands. In this cocktail containing the all the ingredients is heated to 94-95⁰C for 15 to 30 seconds.

2. Annealing: when the temperature is lowered (cooled to 50-65°C) to enable the DNA primers to bind to the template DNA. The exact temperature depends on the melting temperature of the primers.. This step usually takes about 10-30 seconds.

3. Extension: when the temperature is raised (72°C) and the new strand of DNA is made by the Taq polymerase enzyme. 72°C is the optimum temperature for the Taq polymerase to build the complementary strand. Duration of this step depends on the length of DNA sequence being amplified.

These three stages are repeated 20-40 times, doubling the number of DNA copies each time. Therefore if you are design a PCR experiment with normal temperature settings and reagent amounts (primers, dNTPs, polymerase) for 20 cycles. If you want to increase the amount of DNA produced then you have to simply increase the number of few cycle extra. In given options, run 5 extra cycle is the correct answer.

1. You may not get any amplified DNA product if you will simply increase the overall temperature of PCR steps. Suppose temperature higher than the melting temperature then primers will not anneal to the templet DNA therefore there will not be any amplification.

2. Increasing the length of Primers will not produce extra copy of DNA and it will also create nonspecific binding.

3.Doubling the amount of dNTPs will not produce extra copy of DNA.