PCR (in vitro)

Question 1

What is needed to break H bonds?

Answer 1

energy and heat

Question 2

What is needed to polymerise nucleotides?

Answer 2

enzyme DNA polymerase (thermostable) = Taq polymerase

Question 3

When and who by was PCR first initiated?

Answer 3

1985- Karry Mullis

Question 4

Brief

Answer 4

• a specific region of DNA is replicated over and over again, only replicating specific regions of genome/DNA not the whole genome
• billions of copies of particular region can be obtained quickly + cheaply
• creates enough DNA that can be analysed simply.

Question 5

What are the three stages in PCR?

Answer 5

Denaturation
Annealing
Extension

Question 6

Denaturation

Answer 6

95 Degrees Celsius
break hydrogen bonds, strands separated
-two strands of DNA denatured by heat, not enzymatically
-just separates the two strands of DNA, does not destroy
-process is reversible

Question 7

Annealing

Answer 7

55-65 Degrees Celsius
primers anneal to a complementary sequence on template DNA - you need two primers to amplify DNA per DNA.
-DNA allowed to cool in the presence of two primers(synthetic oligonucleotides)
-one primer binds to one strand of DNA, and one to the other
-primers must have 3’OH group, which on the two primers face each other
- temp at which primers bind depends on length and sequence

Question 8

Extension

Answer 8

72 Degrees Celcius
synthesis of new DNA, template strand copied
-DNA polymerase binds to 3’OH of primer and adds dNTPs to synthesise a new strand in 5’ to 3’ direction
-DNA polymerase has to be thermostable
-isolation of Taq polymerase from thermus aquticus
Repeat all steps again about 30x.

Question 9

PCR reaction components

Answer 9

• several components in water
• Tris-HCl (pH 8.3 at 25 degrees celsius) = 10-50mM (buffer)
• Magnesium chloride = 1.2-2.5mM (Mg ions cofactors)
• Potassium chloride = 50mM (ccorrect ionic conditions)
• BSA = 100gml-1 (protein)
• dNTPs = 200M each
• DNA polymerase = 0.5-5U
• Primers = 0.1 - 1M (e.g.synthesise 3 DNA = 6 primers needed)
• Template DNA = 1-long (sample/reference sample)
• salts inluded in pre made buffer

Question 10

Controls

Answer 10

• negative control = no added template, expect to see no products after PCR process, if products present then must be contamination.
• Positive control = standard, good quality template known to work

Question 11

Precautions against contamination

Answer 11

• pre and post PCR processing physically separated
• usually a separate room or a laminar flow hood
• equipment for setting up PCR reactions should be kept separate.
• gloves and gowns worn
• aerosol-resistant tips used.

Question 12

How can you tell if PCR has worked?

Answer 12

• check products on a GEL
• can visualise the DNA
• should be expected size
• should only be one band