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Flashcards in PCR Deck (33)
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1
Q

When was the first publication?

A

1985

2
Q

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

A

Mullis + Smith 1993

3
Q

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

A

3rd

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

4
Q

Uses of PCR:

A
  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
5
Q

Total product of n cycles

A

2^n with start and intermediate

6
Q

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

A

1, priming

7
Q

Total product of PCR-like reactions

A

n

8
Q

PCR-like reactions use _____ amp.

A

linear -> sequencing reactions

9
Q

Temperatures of stages:

A

94-96, 65, 72

10
Q

DNA strand separation is ____ and depends on :

A

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
11
Q

Why is full separation required?

A

To prevent fast snap-back renaturation

12
Q

Constraints of separation process

A
  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
13
Q

Primer annealing takes

A

10-30 seconds

14
Q

How to design an oligonucleotide primer for PCR?

A

Parameters + conditions different for individual PCR strategies

15
Q

In amplification of mouse/human genomic DNA ->

A

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

16
Q

At melting temperature:

A

50% annealed

17
Q

Wallace rule

A

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

18
Q

When would you use the wallace rule?

A

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

19
Q

When would you use the Bolton + McCarthy rule?

A

14-70mers, M+<0.4M

20
Q

Primers and target sequences are not in

A

thermodynamic equilibirum

21
Q

Kinetic considerations of annealing:

A
  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
22
Q

An important side reaction in annealing stage

A

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

23
Q

Hot start

A

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

24
Q

Outline primer extension

A

dNTPd(4 bases) incorporate onto oligonucleotide primed template

25
Q

What’s the duration of primer extension?

A

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

26
Q

What happens if AT< ET

A

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

27
Q

What can we do to alleviate AT

A

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

28
Q

How is mutagenesis achieved?

A

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 .

29
Q

PCR product is measured by

A

DNA binds Ethidium bromide -> fluoresence v cycle #

30
Q

Alternative to hot start

A

Taq antibody. modified enzyme only active from TA

31
Q

Use specific primers to generate a ____ with PCR

A

genomic library

32
Q

If 2 polymerases are used results are improved since

A

better removal of mismatched bases

33
Q

Site-direct mutagenesis:

A

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