Polymerase Chain Reaction (PCR) and Gel Electrophoresis

Question 1

PCR

Answer 1

DNA amplification - produces large sample of DNA from very small sample

Question 2

Process

Answer 2

In thermocycler:
1. DNA sample heated to 95°C to separate DNA strands (denatured)
2. Cooled to 50°C allowing specially manufactured primers to base pair with 3’ ends of each DNA strand
3. Heated to 72°C to activate Taq polymerase which attaches nucleotides to primers, forming new strands
4. Cycle repeated over and over - 1 billion of desired sequence at 30 cycles

Question 3

Gel Electrophoresis

Answer 3

Separates DNA fragments based on size

Question 4

Process - Components

Answer 4

1. DNA stained with negatively charged dye to make samples easier to see
2. Gel of either polyacrylamide or agarose (“filter” with many pores that DNA “weave” through) submerged in buffer (aqueous solution with many salts)
3. Positively charged anode one end, negatively charged cathode other end (where DNA wells are), creating electric current that allows negatively charged DNA to move from negative to positive

Question 5

Process - DNA Movement

Answer 5

4. Shorter strands move faster and further down gel, fragments of same size move together in band
5. One well contains standard DNA with DNA of known length (DNA ladder)
6. After gel run, stained with ethidium bromide which binds to DNA and shows up under fluorescent light
7. Gel run under UV and movement compared to that of DNA ladder to estimate size