PCR Flashcards
When was the first publication?
1985
By who and when was PCR used for site-directed mutagenesis
Mullis + Smith 1993
Which cycle does the desired PCR product begin to appear? and what is this?
3rd
- double-stranded prod. w/o ss template based extensions
Uses of PCR:
- Sequence specific detection (+amp) of DNA
- Generation of DNA for specific applications
- engineering of DNA - mutagenesis +
- Quantitation of DNA -> exp. amplification
Total product of n cycles
2^n with start and intermediate
In PCR- like reactions, only ____ strand amplified due to
1, priming
Total product of PCR-like reactions
n
PCR-like reactions use _____ amp.
linear -> sequencing reactions
Temperatures of stages:
94-96, 65, 72
DNA strand separation is ____ and depends on :
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
Why is full separation required?
To prevent fast snap-back renaturation
Constraints of separation process
- 1 for all + temp. dependency on pH
- has to work well with enzyme -> stable at denaturing end
- evaporation/ condition in reaction vessel
Primer annealing takes
10-30 seconds
How to design an oligonucleotide primer for PCR?
Parameters + conditions different for individual PCR strategies
In amplification of mouse/human genomic DNA ->
16mer used : 4^16 > 3GB base complexity
At melting temperature:
50% annealed
Wallace rule
Td = 2C(A+T) + 4C(G+C)
When would you use the wallace rule?
14-30mers with 1 sequence filter bound 0.9M NaCl
When would you use the Bolton + McCarthy rule?
14-70mers, M+<0.4M
Primers and target sequences are not in
thermodynamic equilibirum
Kinetic considerations of annealing:
- n of target sequence isn’t constant -> as low as 1 in the beginning then exponential increase
- Fast kinetic = large molar excess of primer e.g. 20uM each primer, 1k copies in 50 ul => 10^12 excess
An important side reaction in annealing stage
Self-annealing of primers at 3’ end followed by extension
Hot start
minimises unspecific priming - DNA p added after heat denaturation so mismatched primers can’t form
Outline primer extension
dNTPd(4 bases) incorporate onto oligonucleotide primed template
What’s the duration of primer extension?
long enough to synthesise full length PCR product. e.g. 1mm/kb for Taq
What happens if AT< ET
Primers separate from target at extension temp. + some extension occurs at annealing temp.
What can we do to alleviate AT
Design so AT not much lower than ET or AT= ET
How is mutagenesis achieved?
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 .
PCR product is measured by
DNA binds Ethidium bromide -> fluoresence v cycle #
Alternative to hot start
Taq antibody. modified enzyme only active from TA
Use specific primers to generate a ____ with PCR
genomic library
If 2 polymerases are used results are improved since
better removal of mismatched bases
Site-direct mutagenesis:
Single- base mismatched between amplified primer and template. Incorporate into template sequence -> primer extension
