Increasing Annealing temperatures for BSP-PCR - New Paper in Biotechniques (Feb/16/2007 )

How exactly does introducing one bias overcome another ?

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The way I read the biotechniques paper it says touchdown PCR eliminates bias, not just high annealing temperature...

this would be because at high annealing temperatures all secondary structure has been melted and there is not less efficient amplification for sequences that are hypo-methylated. You want to touchdown so you do not sacrifice your yield.

We have switched to touchdown for all amplifications with BST, just use your regular primers and do PCR 5 cycles each 65, 63, 61, 59, 57, 55... (or whatever pattern you like) then we wrap up with 50 cycles at 50 (yes overkill but at this point because of previous touchdown it should not bias) then we use 10ul of PCR product for each pyrosequencing reaction (is nice because of high yield can sequence with multiple primers from same amplification reaction) Anyway, my 2 cents... Good luck

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Hi beccaf,

thanks for the interesting news about touchdown PCR which are surely worth a trial. However, the paper by Shen et al does not report touchdown PCR but a constantly high annealing temperature of 60°C - that is why I thought about two-step protocols.

Another question - do you purify the PCR product before the pyrosequencing reaction?


Krümel

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Absolutely we use biotinylated primer for one strand then purify with streptavidin beads (there is a purification station with the pyrosequencer) I think this is essential to get good sequencing...

Excerpt from Shen et al...
Consistent with previous results, in all
cases the bias was toward preferential
amplification of unmethylated DNA.
By increasing the annealing temperature
of PCR, we could overcome
PCR bias for all genes, except CDH13.
One condition was carried out by
touchdown PCR for ESR1 and MGMT
genes, 58°, 56°, 54°, 52°C and 60°,
57°, 54°, 51°C, respectively (Figure
3). The purpose of touchdown PCR is
to improve the amplification efficiency
for methylated DNA in the first few
cycles at a relatively high temperature
without decreasing the yield of
PCR amplification as the annealing
temperature drops later. We found that
for MGMT gene, touchdown PCR by
starting the annealing temperature at
60°C for 5 cycles, gradually decreasing
to 57°C for 5 cycles, 54°C for 5 cycles,
and finally to 51°C for 35 cycles
indeed improved the PCR efficiency
for methylated DNA and corrected the
PCR bias completely.

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Sorry, next time I read more thoroughly before posting ... but nevertheless, they report also decreased bias for constant high annealing at 60°C, but also for their preliminary experiments.

Btw - 2-Step PCR also works, but I have not yet checked for the bias.

Krümel

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Please don't apologize, I actually missed that just using 60 worked for certain primer sets, I think it just depends on your mindset when reading the paper (or should I say skimming in my case)... I completely agree that both should work, but I still think I like touchdown because we are able to use the same PCR program for all of the promoters we are analyzing even though the primers have different Tms... TFTP and good luck with your experiments...

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