PCR

Question 1

What is PCR and what is it used for ?

Answer 1

PCR = Polymerase Chain Reaction

Widely used technique to amplify DNA in vitro.

Question 2

When did the first publications concerning PCR occur ?

Which scientists won a nobel prize working on PCR and with whom ?

Answer 2

“First” publications: 1985, but there are earlier suggestions (Khorana group, Kleppe 1971)
Nobel Prize in Chemistry 1993: Kary Mullis for PCR, shared with Michael Smith for site directed mutagenesis

Question 3

What are the 3 formal steps of PCR ?

Answer 3

1) Strand separation of double stranded DNA (
denaturation, melting)
2) Annealing of primers
3) Extension of primers (DNA synthesis by DNA polymerase)

Question 4

How many PCR cycles are required to obtain our first desired dsDNA fragments ?

Answer 4

3 cycles.

Question 5

What are the most common uses of PCR ?

What are these based on ?

Answer 5

• “Sequence-specific” detection (and amplification) of DNA
• Generation of DNA for specific applications (e.g. sequencing)
• Engineering of DNA (mutagenisis and much more) - Quantitation of DNA
  all the above are based on an exponential amplification of DNA (both strand are being amplified).
  Total product count after n cycles: 2^n (including starting sequence and intermediate products)

Question 6

What are PCR-like processes ?

Answer 6

These are processes when only one strand is amplified, due to priming to only one strand total product count after n cycles: n (excluding original strand, if present) therefore also called linear amplification e.g. used for sequencing reactions.

Question 7

At what temperatures do strand separation (denaturation), primer annealing and extension occur ?

Answer 7

Denaturation = 94-96 degrees
Primer Annealing = 65
Extension = 72

Question 8

What were the DNA polymerases known in 1985 ?

What are their properties ?

Answer 8

DNA polymerases known in 1985 included DNA polymerase I (or Pol I) originally purified from
E.coli, but ubiquitous in prokaryotes (1956), involved in DNA repair (and DNA replication) has three main enzymatic activities :
5’->3’ DNA-dependent DNA polymerase, primer dependent, template dependent
3’->5’ exonuclease [relevant for proofreading]
5’->3’ exonuclease [“nick translation”]

Question 9

Why is the 5’->3’ exonuclease activity often unwanted in molecular biology reactions ?

Answer 9

Because the main function of the 5’ to 3’ exonuclease activity is to remove ribonucleotide primers that are used in DNA replication.

Question 10

What is the Klenow fragment, or Klenow polymerase ?

Answer 10

An in-vitro proteolytic treatment of Pol I can remove the domain necessary for 5’->3’exonuclease activity, yielding the Klenow fragment (or Klenow polymerase) –> enzyme used in 1985 publication.

Question 11

What is the [porblem of using the Kelow polymerase for PCR ?

How was this problem overcome ?

Answer 11

Problem : Klenow works best at about 37 degrees –> denaturation requires high temperatures –> Klenow will be deactivated => requires addition of new enzyme at every cycle
Solution : find an enzyme that is heat-stable in PCR 1986/1987 : Taq polymerase isolated from thermophilic bacterium Thermus aquaticus, stability about 30min at 95 degrees, or 9 min at 97.5 degrees

Question 12

What is the problem with using tag polymerase for PCR ?

Answer 12

Taq has no 3’->5’ exonuclease activity = no proofreading

Question 13

What is DNA denaturation dependant on ?

Answer 13

• Salt ion concentrations: higher ionic strength => more stable base pairing
• Base pair composition: G/C more stable than A/T => initially local melting at AT rich
• pH: basic conditions favor stand separation

Question 14

Why is full separation required ?

Answer 14

Otherwise, there is a possibility of very fast snap-back renaturation.

Question 15

What are the main contraints linked to PCR ?

Answer 15

One buffer system for all the PCR steps :
- PCR enzyme has to work well in this buffer
- PCR enzyme should be stable at denaturing conditions
Further technical constraints :
- temperature dependency of buffer pH
- evaporation and condensation in reaction vessel (heated lid, mineral oil cover layer)
- denaturation at 94-96 degrees, appropriate buffer system, salt concentrations

Question 16

What is the most critical/complex step in PCR ?

Answer 16

Primer annealing process.

Question 17

How long would a primer have to be to amplify the entire human/mouse genome (assuming random genome)?

Answer 17

A 16mer, because 4^16 ~ 4,300,000,000 > 3Gbases complexity.

Question 18

What should the melting temperature be ?

Answer 18

It should be such that at the melting temperature, 50% of the sequence has annealed.

Question 19

What is the Wallace rule ?

Answer 19

Wallace rule = a rule for calculating the melting temperature Tm of short oligonucleatides : Td = 2oC(A+T) + 4oC(G+C)
Rule works for 14 - approx 30mers, with one sequence filter bound, at 0.9M NaCl

Question 20

What is the Bloton and McCarthy rule ?

Answer 20

Bolton and McCarthy (modified, e.g. Maniatis):

Tm = 81.5oC + 16.6 (log10[M+]) + 0.41(%[G+C]) - 675/n), works ~ 14-70mers, [M+] <0.4M

Question 21

Give an example of more sophisticated method that can be used to calculate the Tm of an oligonucleotide ?

Answer 21

Nearest-neighbor thermodynamics (software packages).

Question 22

How long should the annealing period usually be ?

Answer 22

Usually short e.g. ~10-30s

Question 23

Why is “hot start” PCR used ?

Answer 23

To minimize unspecific priming + the formation of primer dimers at their 3’ ends, followed by extension.

Question 24

What does DNA Pol require for its activity ?

Answer 24

• an oligonucleotide-primed template
• free dNTPs (dATP, dTTP, dGTP, dCTP)
• divalent cations, usually Mg2+, plus monovalent ions

Question 25

What is the temperature of the extension phase ?

Answer 25

e.g. 68-72oC; usually close to optimal temperature of polymerase (i.e. 75-80oC for Taq)

Question 26

How long should the extension stage last ?

Answer 26

Long enough to synthesize full length of PCR product (e.g. 1min/kb for Taq)

Question 27

What are the potential consequences if the annealing temperature is much lower than the extension temperature ?
What does this impose ?

Answer 27

Primers may separate from target at extension temperature.

Thus, some extension should ready occur at annealing temperature.

Question 28

How could primers be designed for an optimal annealing-extension transition ?

Answer 28

We can design primers so that annealing temp is not much lower than extension temp or design the same temperature for annealing and extension.

Question 29

What is the error rate of Klenow ? - of Taq ?

Answer 29

Klenow : 1/50,000

Taq : 1/2,000 (or worse)

Question 30

What do DNA Pol error rates depend on ?

Answer 30

Reaction conditions :

• [dNTP]
• [Mg2+]

Question 31

What is Pfu ?

Answer 31

Pfu = thermostable DNA Pol found in the hyperthermophilic archaeon Pyrococcus furiosus, has 3’–>5’ exonuclease (proofreading) activity, but is slower that Taq

Question 32

All DNA Pol’s seems to have both advantages and flaws.

What can be envisaged to overcome this problem ?

Answer 32

Add in a mixture of polymerases.

Question 33

Why is blunt end cloning not efficient ?

Answer 33

No sticky ends.

Question 34

What overhangs are produced by Taq ?

How can these be used ?

Answer 34

3’A overhangs –> can be efficiently exploited for TA cloning

Question 35

What overhangs are produced by Pfu ?

Answer 35

NONE (I think).

Question 36

What are the two main ways of generating a restriction fragment from a PCR product ?

Answer 36

1. Cut at site within PCR product –> works only if suitable sites are present
2. Add sites to primers (these sites should be absent in target sequence) –> digestion of the PCR product will generate a DNA with EcoRI and MluI overhangs

Question 37

What are the three steps to indtroduce a mutation via PCR ?

Answer 37

1. Generate two separate PCR products, two external primers, two internal primers with mutated sequence
2. Combine the two PCR product and run new PCR with the two external primers
3. Final product

Question 38

If we start with one copy of our DNA fragment, what is the yield after n cycles ?

Answer 38

2^n

Question 39

If we start with 1ng of target DNA, calculate 40 cycles.

Answer 39

1,100 kg of DNA

Question 40

If we start with 1pg of target DNA, calculate 25 cycles.

What about w/ a cycle efficiency of 1.8 ? - 1.5 ?

Answer 40

Efficiency of 2 : 33.5 ug

• —————- 1.8 : 2.4ug
• —————- 1.5 : 25.5 ng

Question 41

What is the risk of a large number of cycles ?

justify your answer.

Answer 41

Increased error :

a) each final product has undergone a synthesis n times
b) reaction conditions usually conditions deteriorate with increased cycle count