PCR

Question 1

What is PCR?

Answer 1

A pocess that could make unlimited numbers of copies of genes

Question 2

Whar are the requirements for PCR?

Answer 2

• Target DNA
• Two oligonucleotide primers (short single stranded pieces of synthetic DNA)
• dNTPs
• A thermostable DNA polymerase (usually from therus aquatiqus)
• The correct buffer
• A thermal cycling (PCR) machine

Question 3

Describe a typical PCR protocol

Answer 3

1. The mixture is heated to 95^oC for 30 seconds→To denature the DNA
2. The mixture is cooled to 55^oC for 30 seconds→ To anneal the primers to the DNA
3. The mixture is heated to the intermediate temperature of 75^oC (optimum fo DNA polymerase) for 1 minute→ To synthesise new DNA by extending the primers with dNTPs
4. This is repeated for x(30) amount of cycles

Question 4

Name some applications of PCR

Answer 4

1. Straghtforwrd amplification
2. Gene cloning
3. Medical detection (SNPs that cause disease)
4. Criminal detection
5. Antient DNA

Question 5

What are some other uses of PCR?

Answer 5

• Mutagenesis/ site directed mutagenesis:

This is achieved by introducing a mutation in the centre of a primer and it will be incorporated into all copied molecules

• Introduction of novel restriction sites:

You can add what you like to the 5’ end of a primer and it will be copied!

Question 6

Describe reverse transcriptase PCR

Answer 6

1. The mRNA is taken from the cell
2. The mRNA is copied into cDNA
3. Amplify the cDNA by PCR

Question 7

TRUE or FALSE: Quantitative PCR can be used to find out how many genes there are in a cell?

Answer 7

TRUE

Question 8

What can be achieved if RTPCR is used in conjunction with QPCR?

Answer 8

You can find out how much mRNA is being transcribed from a different gene

Question 9

What is inverse PCR?

Answer 9

The primers face outward on a plasmid to allow the entire plasmid to be copied

Question 10

TRUE or FALSE: PCR can be used to create synthetic genes

Answer 10

TRUE

It can also be used to mimic (molecular) evolution

Question 11

Describe the properties of oligonucleotide primers

Answer 11

• The ae made of synthetic DNA
• They are single-stranded DNA (ssDNA)
• Typically~ 18 bases long (should be 16-22 bases)
• Should have as close to 50% G/C content as possible
• Should have a “GC clamp” at he 3’ end (this means that the last two bases of the primer should be any combination of Gs and Cs- GG CC CG GC) ►This means that 3 hydrogen bonds will be present between the bases so the primer will be held more tightly onto the template at the 3’ end (allowing DNA polymerase to latch onto it easier

Question 12

How do primers anneal (hybridise) to the denatured template DNA?

Answer 12

By complementary base pairing

Question 13

Each cycle of PCR doubles the number of copies of DNA: How many copies should be generated in theory byb 30 cycles?

Answer 13

2³⁰= 1.07 x10⁹ copies from each molecule of template DNA (assuming 100% efficiency)

Question 14

Why must the DNA polymerase enzyme be thermostable?

Answer 14

Otherwise it would be denatured at the start of each cycle

Question 15

Which DNA polymerase is commonly used for PCR?

Answer 15

Taq DNA polymerase

• This enzyme catalyses DNA syntheis at 72^oC
• It can withstand 95^oC fo short periods of time

Question 16

What is the main disadvantage of Taq DNA polymerase?

Answer 16

• It lacks proofreading activity
• This is because it has no 3’-5’ exonuclease (cutting out backwards)
• This means it can make mistakes in the new DNA
• However, other polymerases are available