Chapter 10

Question 1

What is PCR?

Answer 1

Polymerase Chain Reaction is a technique used to make multiple copies of a DNA sequence.

Question 2

Describe the process of the first two rounds of PCR.

Answer 2

1. The two DNA strands of the target sequence separate forming sing-stranded DNA.
2. Short primers attach (anneal) to each of the two DNA strands.
3. Taq DNA polymerase extends the two DNA strands by adding complementary nucleotides.
4. End of the first cycle: two DNA strands are formed, identical to the original DNA target sequence.
5. The four DNA strands of the target sequence separate.
6. Short primers attach (anneal) to each of the four DNA strands.
7. Taq DNA polymerase extends the four DNA strands by adding complementary nucleotides.
8. End of the second cycle: Four DNA strands are formed, identical to the original DNA target sequence.

Question 3

How do you calculate the number of strands there will be after a certain number of cycles of PCR?

Answer 3

2 to the power of the number of cycles

Question 4

What are three 3 steps of PCR?

Answer 4

1. Denaturing: 94-96 degrees hydrogen bonds break
2. Annealing: 50-60 degrees primers attach - allow DNA polymerase to bind as it can only bind to a double strand.
3. Extension: 68-72 degrees DNA polymerase joins the complementary nucleotides to the strands from the primers onwards.

Question 5

What is Gel electrophorosis?

Answer 5

Separate DNA strands based on their length.

Question 6

How does Gel electrophoresis work?

Answer 6

Restriction enzymes are used prior to cut the DNA at specific sites.
DNA has a negative charge.
DNA moves away from the the negative terminal once an electric current is applied to the gel.
Smaller lengths of DNA will move more quickly than longer lengths. This will produce a DNA profile.
DNA ladder is run at the same time with known lengths for comparison.
The DNA is visualised.

Question 7

How does DNA sequencing work?

Answer 7

Extract DNA
PCR – need billions of copies of DNA fragment
Denature the fragments - single-stranded DNA
Add primers. These anneal to the start of the fragment
Add DNA polymerase, normal bases (dNTPs), fewer terminating bases (ddNTPs)
ddNTPs are different because they have no OH group and have fluorescent markers unique to the type of base (e.g. C has a red marker)
DNA polymerase begins extension – adding dNTPs and ddNTPs to the single-stranded fragments
Most of the time extension continues, however, occasionally a ddNTP binds and this stops extension for that fragment.
Because there are billions of fragments, every possible length of base sequence is created.
Fragments are sorted from shortest to longest using capillary electrophoresis.
A laser excites the fluorescent marker on the ddNTPs and a sensor detects the colour and send this to a computer which determines the sequence.