PCR

Question 1

What is done in PCR?

Answer 1

Enzymatic replication of DNA without using an organism

Question 2

What are the 3 cycles of PCR?

Answer 2

Denaturation

Annealing

Extension

Question 3

What are the components of PCR?

Answer 3

Target DNA
dNTPS
Taq. Polymerase
Primers
Buffer

Question 4

How primers are made? length? how is it bound to the DNA and start the synth.?

Answer 4

designed & custom synthesized manually or using software

17-30 nt

forward & reverse primers anneal to the antisense & sense strands respectively and DNA synth. occurs in the region b/w the 2 primers.

Question 5

List the requirements for a primer

Answer 5

3’ G or C residue & last 5 bases at 3’ to be G-C at least 40%

G-C contents should be 40-60%

Tm should be 65-75C(Tm of reverse & forward primers should not differ more than 5C from each other)

Question 6

On what the specificity of a primer depends on? why more specific is useful?

Answer 6

Length

Tm - more the higher

Question 7

How to estimate Tm of a primer? recall the equation

Answer 7

using Wallace’s equation

Tm = (Total G+C) x 4 + (Total A+T) x 2

Question 8

What type of DNA polymerase is used for PCR? How it’s obtained?

Answer 8

Taq DNA Polymerase

by Thermus aquaticus

Question 9

List the characteristics of taq polymerase. DNA synth rate? Drawback?

Answer 9

thermostable up to 94C

• no sig. lost of activity
• stable during pcr cycles’

optimum T = 72C
optimum pH = 8.3

Half life - 40mins

synth rate = 35-100 nt/s

Error rate is high. 1/9000 as it lacks 3’-5’ exonuclease activity. low fidelity

Question 10

What does buffer do in PCR?

Answer 10

Provides a suitable chemical env. for taq. pol.

Question 11

List the components in a PCR buffer with their functions.

Answer 11

Tris HCl - to keep the pH 8.3

25mM KCl - K+ stabilize the (-) charge of DNA

Mg2+(1-5mM) - cofactor for taq polymerase and stabilize (-) charges and facilitate primer annealing

gelatin or BSA(albumin) - stabilize taq. pol. and effective when pcr inhibitors are used

Question 12

dNTPs in PCR what amounts are used? What happens if higher concentrations are used?

Answer 12

used in equimolar concentrations

optimum 0.2 mM

increase misincorporations and can inhibit PCR

Question 13

Explain the steps in the PCR procedure

Answer 13

Denaturation
-@ 94C for 30s

Annealing
-T is reduced to 45-72C for 30s. at this T primers are annealed to the DNA

Extension
-T is increased to 72C - optimum for taq polymerase. it extend the annealed primer by addition of nts to 3’ ends of primers

Question 14

What does annealing T depends on in PCR? usually?

Answer 14

on Tm (melting T)
annealing T < Tm
usually annealing T = Tm - 5C
So forward & reverse primers Tms cannot be differ by more than 5C

Question 15

Calculate the number of DNA copies produced in 30 cycles

Answer 15

2^n

Question 16

Usually how long does it take to extend 1kb fragment?

Answer 16

60s

Question 17

What is thermal profile? Draw one.

Answer 17

T vs Time plotted with the state of DNA (ss/ds)

Question 18

How to automate PCR? What are advantages & drawbacks?

Answer 18

using PCR Thermocyclers - have metal blocks that can heat/cool to hold PCR tubes

high accuracy
heats & cools quickly - ramp times are low

but expensive

Question 19

List the applications of PCR?

Answer 19

Cloning of DNA - rapid, cost-effective than traditional methods

Diagnosis
-High sensitivity - even a single DNA can be amplified and detected - viral, bacterial diseases, prenatal diagnosis

DNA sequencing

Forensics

• DNA fingerprinting
• identifying individuals