PCR

Question 1

What does PCR enable?

Answer 1

PCR enables copying and amplification of small and specific regions of DNA (50 bp - 50 kb)

Question 2

What are the steps of PCR and its temperatures?

Answer 2

Denaturation - 95°C
Annealing - 50-60°C
Extension - 72°C

Question 3

Explain denaturation

Answer 3

Double stranded DNA is made single-stranded

Question 4

Explain annealing

Answer 4

Short primers anneal by complementary base pairing

Question 5

Explain extension

Answer 5

New strand of DNA is synthesised

Question 6

What is needed for PCR?

Answer 6

DNA, buffer, dNTPS, DNA pol., Mg2+ ions and primers

Question 7

What are primers?

Answer 7

Short (17-30 bp) synthesised oligonucleotides
Specify region of DNA to be amplified

Question 8

How do you design primers?

Answer 8

Based on prior knowledge of DNA sequence of your region of interest

Question 9

What happens if a primer is too short or too long?

Answer 9

<17 bp = anneal to random places
>30 bp = will not bind easily to correct region

Question 10

Where does the forward primer go?

Answer 10

For the anti-sense strand

Question 11

Where does the reverse primer go?

Answer 11

For the sense strand

Question 12

What does Taq polymerase do?

Answer 12

Most commonly used polymerase
Does not proof read = errors produced
Used for analytical work

Question 13

What does the Vent polymerase do?

Answer 13

Moderate speed and error
Used for amplifying longer fragments

Question 14

What does the Pfu polymerase?

Answer 14

Very low error rate and slow
Used for sequencing and cloning

Question 15

What is a quick tip for primer design?

Answer 15

Forward primer -> identical to sense strand (binds to anti-sense)
Reverse primer -> reverse and complements of sense strand (binds to sense strand)

Question 16

How to identify the optimal annealing temperature?

Answer 16

1. Length -> longer (more interactions with template DNA)
2. GC content -> more (stronger interactions)

Question 17

What happens if Mg2+ [] is low?

Answer 17

Too low = pol. cannot function, no/little product

Question 18

What happens if Mg2+ [] is high?

Answer 18

no denaturation promotes non-specific binding of primers to other DNA regions reduces specificity of pol.

Question 19

What is gel electrophoresis?

Answer 19

Technique used for analysis of DNA and RNA molecules

Question 20

How does gel electrophoresis work?

Answer 20

DNA/RNA of diff. sizes will migrate at diff. speeds through a matrix under an electric field

Question 21

Where are smaller and bigger fragments located in the gel?

Answer 21

Smaller fragments = bottom (fast)
Bigger fragments = top (slow)