Protocol Online logo
Top : New Forum Archives (2009-): : DNA Methylation and Epigenetics

Amplification of bisulfite converted gDNA for sequencing - (Nov/22/2010 )

Greetings fellow scientists!
I am trying to amplify bisulfite converted genomic DNA for subsequent capillary sequencing, but I am experiencing problems. I have tried two different polymerases of which only the Dynazyme produces a band on a gel:

(1) AmpliTaq Gold polymerase
AmpliTaq Gold

(2) DynazymeII Hotstart polymerase
Dynazyme II Hotstart Pol

Does anyone have any insight to why AmpliTaq is not working but the Dynazyme is? I have tried varying reagent concentrations (including primers, template) and thermocycling conditions but to no success.

My other question is this: since I am going to sequence the amplicon (using Big Dye chemistry and capillary sequencing), is there any reason why I shouldn't use the DNA amplified with DynazymeII? From reading lots of papers, I see people using Taq polymerases, but they need the TA ends for cloning (followed by sequencing).

Thanks in advance :)


Edit: The sequencing results are used for methylation analysis of promoter regions.


hi there, different Cation concentration can also affect the PCR which is probably something you are seeing.

for PCR, cloning and then sequencing normal Taq is favoured to allow for TA-cloning into a sequencing vector. Both are hot starts so they should work.