Question

1) 7 pts - Discuss how the technique of PCR allows a scientist to quickly clone a particular piece of DNA. Be clear about the importance of 2 primers and Taq DNA polymerase. Make sure you explain how you do a PCR – what is needed.

2) 6pts - Describe the study we discussed in class where scientists were attempting to assess deletion alleles in patients with muscular dystrophy? Describe what they did and what they found. What about MD allowed them to study it this way?

Answer 1

Ans 1. PCR

Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA region in vitro (in a test tube). PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.

It generates billions of copies of a particular DNA fragment (the sequence of interest, DNA of interest, or target DNA) from a DNA extract (DNA template).

PCR is therefore a technique of purification or cloning.

PCR is based on three simple steps -

1. Denaturation of the template DNA into single strands occurs at 92 ° C.

ii. Annealing of primers to each original strand for new strand synthesis. These primers are complementary to template. This step requires 72 ° C temperature.

iii. Extension of the new DNA strands using 2 complementary primers.

Taq polymerase which is obtained from thermophile bacteria Thermas aquaticus which can easily withstand high temperature required for this step.

Ans 2. Majority of the deletions were located at distal hot spot region that encompasses exons and 14.3% of the deletions were located at the proximal hot spot region (exons 2-19). In this study population, the deletion rate was 71% and was more frequent in the distal end exon.

Multiplex PCR methods allow detection of approximately 98% of deletions, which accounts for 65% of all mutations. Locations of deletions in the dystrophin gene are apparently nonrandom with a preponderance found in two “‘hot spot” regions[5,6] at the 5’ terminus and in the distal half of the central rod domain around exons 44-53.