AMB3 Flashcards
(12 cards)
What are the 3 steps of PCR
dentauring step - 90+ degree to break H bonds between dsDNA
Annealing step - oligo reverse compliment primers 55 degrees ish
polymerisiation step - DNAP recognises dsDNA, 72 degrees
what does temperature required for annealing step depend on?
sequence of primer/target and corresponding mp of hybrid molecule
what is the equation for DNA produced in PCR?
Y=x2^n
Y= DNA produced
X = DNA at the start
n= number of cycles
what could limit dna synthesis causes sigmoidal plateu curve?
taq degradation
dNTP limitation
what are thermal cyclers?
complex incubators that allow temps in reaction samples ot ve varied fast and reproducibly.
Name two enxzymes with 5’-> 3’ exonuclease activity and nick translation?
E,coli DNAP1
Taq
Does taq cause strand displacement?
no
what doe fidelity of dnaps ussually come at the cost of? what is the solution for this?
cost of speed
solution is mixing polymerases
eg. taq is fat bubt less accurate
PFU is slow but more accurate
Describe process of how PCR can be used in pre cloning?
- target sequence in plasmid
- design 2 primers that correspond to adjacent sequence in plasmid but opposite oritentations, one contains desrired mutation. one must be 5’ phos
- PCR amplifcation
- treated with Dpnl
- DNA amplified by PCR remains
- mutated DNA is ligated
- Transformed into e.coli
what does Dpnl do?
RE - recognises G^mATC - cutting only methalyated dA, so plasmid grown in standard E.coli strains will be methylated at these sites.
What is RACE PCR?
Rapid amplification of cDNA ends
used to obtain full length cDNA or extend cDNA beyond currently known fragment
what is RT-PCR?
Real time or quantitative PCR which uses linearity of DNA amplification to determine relative amounts of a known sequence in a sample uses flureoscent dyes etc that intercalete with dsDNA
