MG (PCR)

Question 1

What’s PCR?

Answer 1

• Polymerase Chain Reaction
• method of copying fragments of DNA.
• It is an automated method (rapid and efficient).

Question 2

What things are needed for PCR?

Answer 2

• Small DNA sample
• Taq DNA polymerase
• Primers
• Nucleotides
• Thermo cycler

Question 3

What are the 3 steps in PCR

Answer 3

1. denaturation (95C)
2. annealing (68C)
3. elongation/extension (72C)

Question 4

Outline denaturation

Answer 4

• DNA fragments (sample), primers, DNA polymerase and free nucleotides added to a thermocycler.
• 95°C temperatures cause DNA strands to separate (denature) – hydrogen bonds are broken.

Question 5

Outline annealing

Answer 5

• Primers anneal (join) to their complimentary bases (68°C).
• This provides a starting point for DNA polymerase.

Question 6

Outline elongation

Answer 6

• Temperature increased to 72°C, the optimum temperature of DNA polymerase.
• DNA polymerase attaches free nucleotides to each of the separated DNA template strands.
• The cycle re-starts and the DNA will be amplified (double each time).

Question 7

PCR in more detail…

Answer 7

1. The sample of DNA is mixed with DNA nucleotides, primers and Taq DNA polymerase.
2. The mixture is heated to around 95C to break the H bonds between complementary base pairs and thus denature the double-stranded DNA into two-single strands of DNA.
3. Mixture is cooled to around 68C so that the primers can anneal to one end of each single strand of DNA. This gives a small section of double stranded DNA at the end of each single-stranded molecule.
4. Taq DNA polymerase binds to the end where its double stranded, 72C is optimum temperature.
5. Temp is raised to 72C and this keeps the DNA as single strands.
6. Taq polymerase catalyses the addition of DNA nucleotides to the single-stranded DNA molecules, starting at the end with the primer and proceeding in the 5’ to 3’ direction.
7. When Taq DNA polymerase reaches the other end of the DNA molecules then a new double strand of DNA has been generated.
8. The whole process begins again and is repeated for many cycles.
9. The amount of DNA increases exponentially 1,2,4,8,16,32,64,128 and so on.

Question 8

Advantages of PCR (in vitro cloning )

Answer 8

• Very rapid
• Does not require living cells

Question 9

State some applications of PCR

Answer 9

• Detection of oncogenes
• Detecting mutations
• Forensic science
• Tissue typing
• Monitoring the spread of infectious disease
• Identify viral infections
• Research

Question 10

How can PCR be useful in forensic science?

Answer 10

Small quantities of DNA can be amplified to identify criminals or ascertain parentage

Question 11

How can PCR be useful in tissue typing?

Answer 11

Donor and recipient tissues can be matched to reduce the risk of rejection

Question 12

How can PCR be useful in detection of oncogenes?

Answer 12

Detect the type of mutation leading to cancer. Can inform medication

Question 13

PCR in terms of detecting mutations?

Answer 13

Detecting genetic diseases. Parents may do this before conceiving

Question 14

How can PCR be useful in identifying viral infections?

Answer 14

Verify the type of viral infection present (HIV, hepatitis C infections)

Question 15

How can PCR be useful in monitoring the spread of infectious disease?

Answer 15

helps in monitoring the emergence of new strains

Question 16

How can PCR be useful in research?

Answer 16

Amplifying DNA from extinct organisms e.g. mammoth