PCR

Question 1

What are the applications of PCR?

Answer 1

DNA cloning, DNA sequencing, DNA based phylogeny, functional gene analysis, hereditary disease diagnosis, genetic fingerprinting etc.

Question 2

What equipment is required for PCR?

Answer 2

A thermalcycler, a micro centrifuge or plate, DNA template that contains the DNA you wish to amplify, 2 primers, a thermostable DNA polymerase, dNTPs, buffer solution and bivalent or monovalent cations.

Question 3

Describe the steps in PCR?

Answer 3

Denaturation: 95oC for 2-5 mins to denature dsDNA to ssDNA
Annealing: 55oC to anneal primers to template strand
Extension: 72oC using thermostable DNA polymerase eg. Taq with free dNTPs
Repeat 25-40 times
Long extension at the end to ensure all sequence are complete.

Question 4

What do you do with the products of PCR?

Answer 4

Perform agarose gel electrophoresis to check the purity and quantity.

Question 5

Describe the primers that are used in PCR?

Answer 5

3’ complements, Tm of 55oC, no long repetitive stretches, not compliments, 50% GC content, can have 5’ tag or restriction site dependent on required use, 20-30 nucleotides in length

Question 6

What is the reason for carrying out PCR?

Answer 6

To exponentially amplify DNA