Question

Why do we want/need the presence of Mg2+ in a PCR but do not want it present at all other steps of processing/analyzing a DNA sample? (2 marks)

Identify and explain what generally occurs in each of the 3 main steps of a polymerase chain reaction. (6 marks) 8. Identify and explain what the 3 different PCR controls inform you about the results of a PCR. (3 marks)

Highlight the 2 advantages and 3 disadvantages of using PCR. Ensure to discuss how/why each of these points is beneficial or detrimental. (5 marks)

You have a sample with a stock concentration of 73 ng/uL. Assuming it has a nuclear genome size of 2.3*109 bp and a mitochondrial genome size of 18kbp, to what should you dilute the DNA to if performing a PCR with 2uL DNA and: a. You are amplifying a nuclear loci and wish to add 2500 copies of nDNA (answer in ng/uL) (2 marks) b. You are amplifying a mtDNA loci and wish to add 2500 copies (answer in pg/uL) (2 marks)

For the same reactions as stated in Q10: a. How many molecules of dNTPs would you need to add to each PCR if the nDNA amplicon was 385 bp and the mtDNA loci was 665 bp (assume the PCR plateaus at 1012 copies)? (2 marks) b. If you are adding 2 uL of 0.3uM dNTP to your nDNA reaction, are you adding enough dNTPs so that it is not the limiting reagent? Demonstrate your answer mathematically (3 marks)

You recently extracted a 25mg sample of beef tissue. Quantifying your extracted DNA with PicoGreen, you determine that your stock DNA concentration is 125ng/uL. If you want to amplify two markers, one nuclear and one mitochondrial, where you wish to add a total of 5 ng DNA and 500 pg DNA to each reaction respectively, determine the steps required to make dilutions of your stock sample to achieve the satisfactory concentration of DNA for both reactions. NOTE: the minimum volume you should pipette is 2uL, which is the same volume of DNA you are adding to the PCR. (5 marks)

Answer 1

Answer 1

Magnesium in MgCl2 act as a co-factor for DNA polymerase. Taq DNA polymerase is a magnesium-dependent enzyme hence the optimum concentration of magnesium is for PCR. There are some components in the reaction mixture like template concentration, dNTPs and (EDTA) or proteins may reduce the amount of free magnesium present thus decreases the efficiency of Mg. This leads to poor amplification of PCR.

Mg is a co-factor for Taq polymerase enzyme which is required in PCR for amplification hence Mg is only required in PCR and not other steps or process.

Answer 2

Controls in PCR

1. No-template control / Negative control

A no-template control (NTC) helps us to detect the contamination of the PCR reagents. An NTC reaction contains all components but no template. If positive amplification is detected then it indicates the presence of contaminating nucleic acids.

2. Positive control

A positive control that gives positive amplification in PCR. Nucleic acid from an established cell line and a plasmid containing cloned sequences are commercially available as positive controls. . A positive control is used to test the presence or absence of a target.

3. Internal controls

An internal positive control is used to test for the presence of PCR inhibitors. The duplex reaction is carried out, where the target sequence is amplified and also the one primer-probe set and a control sequence are amplified with a different primer-probe set. If the internal, positive control is amplified but the target sequence is not amplified then this indicates that the amplification is successful but the target sequence is absent .