PCR

Question 1

When was the first publication?

Answer 1

1985

Question 2

By who and when was PCR used for site-directed mutagenesis

Answer 2

Mullis + Smith 1993

Question 3

Which cycle does the desired PCR product begin to appear? and what is this?

Answer 3

3rd

- double-stranded prod. w/o ss template based extensions

Question 4

Uses of PCR:

Answer 4

1. Sequence specific detection (+amp) of DNA
2. Generation of DNA for specific applications
3. engineering of DNA - mutagenesis +
4. Quantitation of DNA -> exp. amplification

Question 5

Total product of n cycles

Answer 5

2^n with start and intermediate

Question 6

In PCR- like reactions, only ____ strand amplified due to

Answer 6

1, priming

Question 7

Total product of PCR-like reactions

Answer 7

n

Question 8

PCR-like reactions use _____ amp.

Answer 8

linear -> sequencing reactions

Question 9

Temperatures of stages:

Answer 9

94-96, 65, 72

Question 10

DNA strand separation is ____ and depends on :

Answer 10

reversible,

• high ionic strength -> bp more stable
• base pair composition: G/C > A/T
• > initial local melting at AT rich
• pH: basic conditions favour strand separation

Question 11

Why is full separation required?

Answer 11

To prevent fast snap-back renaturation

Question 12

Constraints of separation process

Answer 12

1. 1 for all + temp. dependency on pH
2. has to work well with enzyme -> stable at denaturing end
3. evaporation/ condition in reaction vessel

Question 13

Primer annealing takes

Answer 13

10-30 seconds

Question 14

How to design an oligonucleotide primer for PCR?

Answer 14

Parameters + conditions different for individual PCR strategies

Question 15

In amplification of mouse/human genomic DNA ->

Answer 15

16mer used : 4^16 > 3GB base complexity

Question 16

At melting temperature:

Answer 16

50% annealed

Question 17

Wallace rule

Answer 17

Td = 2C(A+T) + 4C(G+C)

Question 18

When would you use the wallace rule?

Answer 18

14-30mers with 1 sequence filter bound 0.9M NaCl

Question 19

When would you use the Bolton + McCarthy rule?

Answer 19

14-70mers, M+<0.4M

Question 20

Primers and target sequences are not in

Answer 20

thermodynamic equilibirum

Question 21

Kinetic considerations of annealing:

Answer 21

1. n of target sequence isn’t constant -> as low as 1 in the beginning then exponential increase
2. Fast kinetic = large molar excess of primer e.g. 20uM each primer, 1k copies in 50 ul => 10^12 excess

Question 22

An important side reaction in annealing stage

Answer 22

Self-annealing of primers at 3’ end followed by extension

Question 23

Hot start

Answer 23

minimises unspecific priming - DNA p added after heat denaturation so mismatched primers can’t form

Question 24

Outline primer extension

Answer 24

dNTPd(4 bases) incorporate onto oligonucleotide primed template

Question 25

What’s the duration of primer extension?

Answer 25

long enough to synthesise full length PCR product. e.g. 1mm/kb for Taq

Question 26

What happens if AT< ET

Answer 26

Primers separate from target at extension temp. + some extension occurs at annealing temp.

Question 27

What can we do to alleviate AT

Answer 27

Design so AT not much lower than ET or AT= ET

Question 28

How is mutagenesis achieved?

Answer 28

Mutation can be introduced anywhere

a) generate 2 separate PCR products, 2 external primers, 2 internal primers with mutated seq.
b) combine 2 PCR product + run new PCR with 2 external primers .

Question 29

PCR product is measured by

Answer 29

DNA binds Ethidium bromide -> fluoresence v cycle #

Question 30

Alternative to hot start

Answer 30

Taq antibody. modified enzyme only active from TA

Question 31

Use specific primers to generate a ____ with PCR

Answer 31

genomic library

Question 32

If 2 polymerases are used results are improved since

Answer 32

better removal of mismatched bases

Question 33

Site-direct mutagenesis:

Answer 33

Single- base mismatched between amplified primer and template. Incorporate into template sequence -> primer extension