PCR and genetic engineering

Question 1

Who developed PCR?

Answer 1

Kary Mullis

Question 2

When was PCR first developed?

Answer 2

1983

Question 3

What does PCR stand for?

Answer 3

Polymerase Chain Reaction

Question 4

What does PCR do?

Answer 4

A technique in which a sequence of DNA can be cloned or amplified

Question 5

Complete the sentence.

DNA is amplified entirley… in an automated system involving ….

Answer 5

1. in vitro

2. Thermocycler

Question 6

What is the general principle of PCR?

Answer 6

General principle: heat stable DNA polymerase extends short ss synthetic primers using DNA as a template
During 30-40 cycles of denaturation,
annealing of primers and extension by polymerase the amount of target DNA doubles with each cycle
After cycles complete many millions of copies generated
Amplicons can then be analysed, typically with gel electrophoresis
Use of automated thermocycler capable of heating and cooling reaction tubes in a very short time

Question 7

What is required for PCR?

Answer 7

Required for PCR:
Source DNA. Only need very small 
	amount; even ancient DNA can be amplified
Supply of nucleotides
Heat-resistant DNA polymerase
Primers for DNA synthesis

Question 8

Give some information about primers. What are they? How long are they?
What do they do?

Answer 8

DNA Primers are short (10-20bp) synthetic strands of ssDNA (oligonucleotides)
The primers are used to initiate DNA synthesis of chosen DNA segment
Primers are complementary to the DNA sequences at the 3’ ends of the region to be amplified I.e. the primers ‘flank’ the target DNA sequence. Forward and reverse primers
Therefore the primers thus determine the segment to be amplified (need to know target sequence)
Primers are always added in excess to the reaction tube and under the right conditions anneal to the complementary sequences flanking the target DNA sequence

Question 9

PCR is based on a heat-stable DNA polymerase, what is the name of this?

Answer 9

Taq polymerase

Question 10

Where was Taq polymerase originally isolated from?

Answer 10

Thermus aquaticus

Question 11

Why is Taq polymerase used for PCR?

Answer 11

Taq polymerase doesn’t denature at high temperatures that are needed to separate the DNA strands at the start of each PCR cycle.

Question 12

Describe the process of Taq polymerase in PCR

Answer 12

Taq extends the annealed primers by adding nucleotides to the 3’ end thus copying the target DNA sequence
The nucleotides (all four dNPT’s) are present in the reaction mixture in excess

Question 13

What are the 3 steps involved in PCR?

Answer 13

1. Denaturation
2. Annealing
3. Extension

Question 14

Describe what happens during the first stage of PCR?

Answer 14

The reaction mixture containing DNA, primers, Taq polymerase and dNPTs is heated to typically 94°C
The ds DNA splits open to ss DNA
This allows ssDNA to now be accessible to ss primers
The heat-resistant Taq polymerase is not denatured

Question 15

Describe what happens in the second stage of PCR?

Answer 15

Temperature reduced to 54-60°C to allow annealing of primers
Primers anneal (bond) to complementary sequences on the template strands
Forward and reverse primers
Once annealing occurs, polymerase-driven extension can begin

Question 16

Describe what happens in the third stage of PCR

Answer 16

Once primers are annealed, temp raised to 72°C
At this temp the Taq polymerase can extend each primer by adding nucleotides present in the reaction mixture to the 3’ end of each primer
Complementary bases are added as the Taq moves along the template strand
Both denatured DNA strands are copied

Question 17

What happens at the end of PCR

Answer 17

The newly synthesised strands act as templates during subsequent cycles
The cycle of denaturation, annealing and extension is repeated over and over again
At the end of cycles an elongation step of 5min to allow completion of any unfinished template strands

Question 18

How long does each PCR cycle take?

Answer 18

4 minutes

Question 19

What does errors in copying mean?

Answer 19

There is a limit to the number of “good” copies

Question 20

What are the advantages of PCR?

Answer 20

1.so specific and powerful that only need tiny quantity of source DNA; DNA can also be impure or degraded
2.Can be used to amplify a gene prior to further cloning in bacterial cells
3.DNA from almost any source can be amplified:
DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders
Detection of viral DNA from cells infected with difficult-to-detect viruses e.g. HIV

Question 21

What could PCR potentially be used for?

Answer 21

PCR can be used to amplify fragments of ancient DNA e.g. dodo DNA or DNA from 40K year-old frozen wooly mammoths
Use in forensics to amplify tiny amounts of DNA from blood, hair or semen found at crime scenes
Only about 20 cells are necessary in a tissue sample for PCR to amplify sufficiently
Use in DNA fingerprinting – each individual will have a different profile
For this purpose the selected region for amplification is highly polymorphic (i.e. vary at high frequency within the population
No two individuals will have the same-sized DNA fragments