Outcome 3

Question 1

What is PCR and what does it enable

Answer 1

Polymerase chain reaction - it enables the amplification of DNA fragments through repeated rounds of DNA synthesis

Question 2

What are the requirements for PCR

Answer 2

Target DNA
Heat stable polymerase
Primers
dNTPs

Question 3

Name the 3 stages of PCR

Question 4

Describe the denaturation stage of PCR

Answer 4

The mixture of target DNA, heat stable DNA polymerase, dNTPs and primers are heated to 95C breaking the hydrogen bonds between bases of the dsDNA and seperating the dsDNA into ssDNA

Question 5

Describe the annealing stage of PCR

Answer 5

THe mixture is cooled to 45-60C and primers which are present in excess bind via complementary base pairing to their recognition sites on the ssDNA. The recognition sites must be located at the 3’ ends that flank the target DNA sequence.

Question 6

Describe the extension stage of PCR

Answer 6

The mixture is heated to 72C, at this temperature the taq polymerase uses the primer as its starting point for DNA synthesis. It catalyses the formation of phosphodiester bonds between the primer 3OH and the 5’ phosphate group of a dNTP that is complementary to the corresponding nucleotide of the target DNA to which the primer is bound.

Question 7

What is meant by exponential amplification

Answer 7

PCR typically consists of 25-30 rounds. Each round produces double the amount of target DNA (exponential).

Question 8

How can we confirm that PCR has worked

Answer 8

By agarose gel electrophoresis as the amplified target DNA should be of a defined fragment size.

Question 9

What sets automated PCR apart from older PCR procedures

Answer 9

Thermal cyclers and taq polymerase.

Question 10

What is a thermal cycler

Answer 10

A fully automated machine that can perform a round of PCR in about 5 minutes.

Question 11

What is taq polymerase

Answer 11

A heat resistant polymerase with an optimum temp of 75-80C

Question 12

What are primers

Answer 12

single stranded oligonucleotides that can bind to DNA through complementary base pairing and act as an initiation site for DNA polymerase to begin DNA synthesis

Question 13

Why are 2 different primers used in PCR and what are they called

Answer 13

Forward primer - anneals to antisense strand

Reverse primer - anneals to sense strand

Question 14

What are universal primers

Answer 14

Primers which are have a common nucleotide sequence complimentary to the base sequence of a particular set of DNA molecules. They can bind to a variety of DNA templates.

Question 15

WHat are specific primers

Answer 15

Primers that bind specifically to a sequence of bases found at the 3’ end of a fragment of DNA of interest.

Question 16

What factors is primers selection based on

Answer 16

Primer length
melting temperature
specificity
complementary primer sequences
base composition

Question 17

Why is primer length a factor in primer selection

Answer 17

Specificity is partly based on primer length
If a primer is too short? non-specific binding possible
If a primer is too long? possible primers anneal to each other.

Question 18

What is the typical range of primer length

Answer 18

15-24 bases

Question 19

Discuss melting temperature with relation to primer selection

Answer 19

Melting temperature = temp at which half of the primer have annealed to the target DNA
BOth forward and reverse primers should have similar melting temperature that is above 60C

Question 20

DIscuss complementary primer sequences

Answer 20

Primers should have low homology - low intra-primer complementarity to avoid hairpins
Primers should have low inter-primer complementarity to avoid dimers

Question 21

WHat is ideal base composition for primers

Answer 21

40-60% GC

Question 22

WHat factors decrease PCR efficiency

Answer 22

Contamination
Incorrectly selected primers
DNA polymerase

Question 23

What steps can be taken to avoid PCR contamination

Answer 23

Set up PCR reaction and DNA analyses should take place in different rooms of lab
Dedicated pipettes are required for PCR, the pipettes should be bleached regularily
Gloves and lab coat should be changed after each stage of PCR

Question 24

What is real time PCR

Answer 24

Based on conventional PCR, instead of analysing the final DNA yield with agarose gel electrophoresis. The DNA is quantified during the PCR reaction using fluouresence. The flourescence is acheived by either fluorescent dye which intercalates between bases or dye + labelled DNA probe which are oligonucleotide linked dyes. The flouresence intensity is measured after each cycle.

Question 25

What are the advantages of PCR

Answer 25

As long as the nucleic acid sequence of the target genome is known, primers can be designed to amplify sections of it.
Sample preparation is automated
It provides results very quickly
It can be used to give a quantitative result (real time PCR)

Question 26

What are the disadvantages of PCR

Answer 26

Cross contamination of samples from pipettes etc can give false positives unless strict decontamination procedures are employed.

Impurities in sample can interfere with PCR, sometimes purification of the clinical sample is needed