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Amplify long fragment from bisulfite treated template - for bisulfite sequencing (Jan/27/2005 )

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I am able to very successfully get small (150-200 bp) products using a nested PCR strategy on bisulfite modified DNA; I do not see products from the larger primers but they are certainly there since the nest works so well. I'd love if I could sequence those larger products (400-500 bp). Does anyone have any hints to get a visible PCR product? Can I just ramp up the cycles? (I do 35 cycles and an MgCl2 concentration of 3 mM) Before you tell me this is just a normal PCR problem, you should know that most papers specify that a nest is necessary, one should not expect to see the larger product, and larger products after modification are fragile and difficult to get.




Hey Labtechie,

I have been able to successfully amplify and sequence 1kb from bisulfite treated DNA.

A nested PCR strategy is the best option. I typically design a hemi-nested primer set that is, I pick a primer pair (A and cool.gif that amplifies around 900-1.2kb in length and then a third primer © within the pair.

So this would require two rounds of PCR amplification. In the first round of PCR I would use Primer A and B, take 1uL of this into a second round of PCR with primer C and either A or B depending on how you have designed things.

If this fails a magnesium titration will do the trick. Again with the hemi nested PCR I would titrate MgCl2 at 0.5mM final concentration intervals.

so I typically do 0.5, 1, 1.5, 2, 2.5 and 3mM.

good luck techie!

Nick laugh.gif


Hi Nick,

I am interested in knowing what cycle number you use for the first and second round PCR and how long you treat your DNA (overnight or shorter).

Thank you.


QUOTE (pcrman @ Jan 27 2005, 04:25 PM)
I am interested in knowing what cycle number you use for the first and second round PCR and how long you treat your DNA (overnight or shorter).

Hi pcrman,

the cycle number I use is 30 cycles for each round of PCR.

Here are my cycling conditions:

95C 4 minutes denaturation followed by 5 cycles of :

95C 30sec (denaturation)
annealing for 90sec (annealing temperature set at 2 degrees below calculated Tm)
72C for 120sec extension.

and then 25 cycles of:

95C 30sec (denaturation)
annealing for 90sec (annealing temperature set at 2 degrees below calculated Tm)
72C for 90sec extension.

Final extension period at 72C for 4 minutes and then a hold at 4C.

This is repeated for the second round of PCR.

I am unsure how you get the degrees symbol so I have omitted it.

The bisulfite treatment was overnight at 50C in the dark, and desalted with Wizard Columns.....

I have also used Human Genetic Signatures' MethylEasy Kit with the same success. The kit is certainly much easier than the conventional method.

I have used AmpliTaq from Applied biosystems and PCR mastermix from Promega and they both perform very well with bisulfite PCR in my hands.


Nick cool.gif


Thanks for sharing. Very impressive.


Hey Nick,

Quick question for you. When you amplify larger 1kb + bisulfite treated products do you still use promega standard PCR master mix? or do you modify it with higher dNTPs/taq etc. to account for your longer amplification times?




the above conditions should be suitable for products greater than 1kb also, but increasing the extension time wouldn't hurt, I did not use a different taq mix than that of the standard. We us promega master mix and that works fine!

Good luck



While I have no experience with bisulphite conversion templates, I do know that very low GC content templates require lowered extension temperatures. With long runs of T's and A's (25 or so is enough) internal to the amplified fragment, the PCR enzyme fails to bind to the opposite strand at common extension temperatures. Since all of your C's are being converted to T's in the bisulphite reaction, the expected GC content of your template is reduced by a factor of two. Long stretches of TA containing fragments seem inevitable. I run such reactions with extension temperatures of 64-66.


Su XZ, Wu Y, Sifri CD, Wellems TE (1996) Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucl Acid Res 24(8):1574-5.


thanks for hte tip phage, I will certainly look into that.



To re-emphasize this point, I just did my first bisulfite conversion PCR this evening, and it worked -- I used an extension temperature of 66C with Qiagen Quantitect QPCR Sybr green mix in a real time cycler. The melting temperature of the products that resulted were 69C for one, and 70C for the other. I'm pretty sure that if I had run the reaction with a 72C extension that it would not have worked.


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