Polymerase Chain Reaction

Question 1

THERMAL
CYCLER

Answer 1

1. InitialDenaturation
2. Denaturation
3. Annealing
4. Extension
5. Final Extension

Question 2

(for Conventional
PCR only)
=to check forPCR
products
(amplicons)

Answer 2

Gel Electrophoresis

Question 3

Essential
components
of PCR

Answer 3

• Taq DNA polymerase
• Primers
• dNTPs
• Buffer
• DNA

Question 4

• A small amount of the DNA to be
  amplified is needed. It contains the
  target sequence.
• May check purity of DNA
  using spectrophotometer
• OD260 and OD280 (Absorbance)

Answer 4

TEMPLATE DNA

Question 5

• 1.8-2.0 ratio=pureDNA
• Lower=protein andsolvent
  contamination
• Higher=RNAcontamination

Answer 5

• Get theratio:

Question 6

• Provide 3’-OHends
• For attachment ofincoming
  nucleotides (dNTPs)
• 0.1-0.5µM in each
  reaction

Answer 6

PRIMER (F-R)

Question 7

• 18-25 (better if <30)
  nucleotides inlength,
  longer=higher Tm
• Tm=52-58 deg C
  GC clamps the primer and
• Ideal GC content=40-60%
  template=increases priming

Answer 7

PRIMER

Question 8

higher
GC%, higherannealing temp (Ta)

Answer 8

Ideal GC content=40-60%,

Question 9

should not
have complementary bases
within the sequences to
prevent formation of hairpin
loop

Or complementarysequences
between F primers and R
primers

Answer 9

Each primer

Question 10

Typically200-250 µM of each
dNTP
- <200µM
* high fidelity:lowyield
>200µM low fidelity (inhibitory)

Answer 10

dNTPs

Question 11

Taq DNA Polymerase(72-74°C)

Answer 11

DNAPolymerase

Question 12

Optimum temp is72°C
Makes an error inevery
125,000th nucleotides
Typically 0,02-0.03 U/µLin
a reaction mixture

Answer 12

Thermus aquaticus

Question 13

• Pyrococcus furiosus
• Has proofreading activity
• High fidelityDNApolymerase
• Makes error in every
  676,000th nucleotide
• Toleratetemp up to95°C

Answer 13

Pfu DNA Polymerase(72-74°C)

Question 14

• MgCl2=cofactor forTaqPol
• Stabilizing agent for annealing
  (reduces repulsion oftemplate and
  primer)
• 0.5-5.0 mM optimal
  concentration

Answer 14

Divalent Cationsand
Reaction Buffers.

Question 15

• Tris-HClbuffer
• Regulates amount
  of H+ ions inthe
  s o l u ti o n:
• 8.0 - 9.5

Answer 15

PCR BUFFERS

Question 16

• must be stored
  in small aliquots to reduce
  contamination. If one aliquot
  becomes contaminated,itis easy to
  discard and start with a newaliquot.

Nuclease may contaminate the
mixture and degrade DNA including primers,template,and PCR product
-

Answer 16

NUCLEASE-FREE
WATER

Question 17

▪A typical PCR incudes _____ of amplification, with a
final extension cycle of 10 minutes at 72°C, followed by
incubation at 4 °C

Answer 17

25-40 cycles

Question 18

The PCR tubes may remain at ____ until the next step such
as running in agarose gel electrophoresis is ready

Answer 18

4°C

Question 19

Initial denaturation

Answer 19

95’C, 5 min

Question 20

Denaturation

Answer 20

94’C, 1 min

Question 21

Annealing

Answer 21

55’C, 1 min

Question 22

Extension/Elongation

Answer 22

72’C, 1min

Question 23

Final extension

Answer 23

72’C, 1min modified

Question 24

Hold

Answer 24

4’C to infinity

Question 25

When____are used, the PCR cycling conditions must be optimized.

Answer 25

New primers

Question 26

When optimizing, you have to consider the factors that affect PCR outcomes.

Answer 26

1. Primer designs 2. Concentration of Magnesium salts 3. Annealing Temp(Ta) 4. dNTPs