2. PCR

Question 1

Define polymerase chain reaction

Answer 1

• PCR is an enzyme based reaction that aims to amplify segments of DNA using DNA dependent polymerase, in a cyclical fashion (30-40 cycles)

Question 2

** How is the hybridization in PCR made specific?

Answer 2

• When primers hybridise with a template, the specificity of the hybridization depends on whether or not the primer is complementary
• There will be no mismatches if the hybridization occurs at the temperature equal to the Tm of the primer = high stringency

Question 3

How is the segment that is to be amplified selected?

Answer 3

• To amplify a segment, the primer must be complementary to the end of the sequence
• In exponential amplification, two primers are needed which are complementary to either end

Question 4

What enzyme is involved in PCR?

Answer 4

DNA dependent polymerase

Question 5

What is annealing?

Answer 5

• Annealing refers to the hybridization of primers
• Both annealing & renaturation are competitive processes but the excess of primers favours renaturation.
• Annealing is more favourable based on the kinetics

Question 6

What are the three properties of DNA Dependent DNA Polymerase?

Answer 6

1. It synthesises new nucleic acids by copying a molecule of DNA
2. It elongates the strand by adding nucleotides to the 3’ end
3. It cannot copy or make RNA which is why reverse transcriptase is needed
   - Reverse transcriptase converts RNA into cDNA

Question 7

What 5 things does PCR need?

Answer 7

1. A ss or ds template
2. Primer
3. deoxynucleotide triphosphates (dNTPs)
4. Mg 2+ ions
5. A buffer with a roughly neutral pH

Question 8

What are the three transition states for PCR?

Answer 8

1. Denatured
2. Annealing - formation of initiating complex
3. Native - optimal pH for elongation

Question 9

Define thermostability & why is it a key property of DNA Dependent polymerase?

Answer 9

• Thermostability refers to the ability to retain activity or resist any physical or chemical changes upon an increase in temperature
• PCR involves multiple rounds of heating so it’s important for the DNA polymerase to be heat-resistant.
• The DNA polymerase Taq polymerase is taken from a thermophillic bacterium such as “thermus aquaticus”

Question 10

What are the steps of PCR?

Answer 10

1. DENATURATION - The sample is heated to 94-96 degrees for about 30s which allows the H bonds to break, forming two single strands
2. ANNEALING -The sample temperature is lowered to 55 degrees for 30s which allows the primer to anneal to complementary sequences
3. ELONGATION - Temperature is raised to 72 degrees to allow Taq polymerase to bind to each priming site & elongate the molecule in from the 3’ end
   - 95, 55, 72

Question 11

• What determines the kinetics of PCR?

Answer 11

• The reaction is dependent on the depletion pf the reactants.
• After multiple rounds, the reaction becomes rate inhibited as it involves acidification due to H+ and dNTPs being depleted

Question 12

Give three applications of PCR in diagnostics

Answer 12

1. Identifying the difference between influenza & swine flu
2. Detecting the presence of TB in sputum
3. Identifying individuals positive for PCR

Question 13

What are two forms of real-time PCR?

Answer 13

1. High resolution melting (HRM) -

2. Probe based version qPCR or allelic discrimination

Question 14

What are two applications of SNP detection by PCR?

Answer 14

1. Antibiotic resistance testing

2. Identifying gene markers e.g cancer

Question 15

What are STRs?

Answer 15

• Short tandem repeats are between 2-5 bases or more

- They are highly polymorphic meaning that they vary between individuals

Question 16

How can be PCR be used to modify & manipulate DNA?

Answer 16

1. Involved in recombinant DNA technology

2. Introducing mutations into DNA sequences