PCR help

Alok Adholeya aloka at teri.res.in
Mon Jun 8 00:06:25 EST 1998


Dear All,


I have a query on PCR fingerprinting. Conventionally during
standardization of PCR we adjust the Mg++ concentration, standardize
annealing time/ temperature, add specificity enhancers like formamide
etc.... . I wrote a small programme for my REP on my PCR Machine.
Instead of complete smears on my gel (which I have been getting) I
ended up having distinct and beautiful finerprint. The programme is as
following:

#1 94 deg C for 2 min
#2 94 deg C for 50 sec
#3 35 deg C for 1:20 min
     Increment of 0.5 deg C per cycle
#4 72 deg C for 2:30 min
#5 GoTo step number 2, 18 times
#6 94 deg C for 50 sec
#7 44 deg C for 1:20 min
#8 72 deg C for 2:30 min
#9 GoTo step number 6, 17 times
#10 4 deg C for 5 min
#11 END

Rationale behind this programme? 
We start with (very) low stringency as the Tm of both the REP primers in
50 mM Na+ ions are 43 deg C thereby allowing even weaker binding
sites have some access to the primer. As the reaction progresses the
stringency is incresed (0.5 deg C per cycle) and within a few cycles
(10) the annealing temperature comes close to Tm. After a while the
majority of the reacton is carried out on extremely highly stringent primer
annealing temperature (one deg above Tm). Extension slightly above Tm
is possible from sties having very accurate match.
The advantage of such a STEP-UP PCR (I call this so because it is
analogous to Touch Down PCR) is that not a great deal of
standardization is involved, no need to titrate Mg++ and other additives.

Then what's the problem?
I have not seen such a protocol for PCR fingerprinting, especially when
the primer used has been extensively studied. Would people accept this
kind of an amplification parameter. If not, why not? Afterall we are using
the machine to enhance specificity rather than conventional PCR
additives. I addend nothing except what was mandatory ie., Buffer,
dNTP, primers, template Taq and water.

Thanks
Alok

Please email me at  : aloka at teri.res.in




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