Protocol Online logo
Top : New Forum Archives (2009-): : General Lab Techniques

DNA sequencing for GC rich sequence - (Apr/13/2014 )

Hello,

 

I have always wondered, when I submit DNA for sequencing, the company asks me to mark if it is GC rich or not. How does this affect the sequencing? What do they do different for GC rich sequences?

-luckyjade-

GC rich sequences bind more tightly to each other than AT rich sequences, because of the binding properties of the guanine and cytosine molecules.  Sequencing these sequences can be difficult, but some of the problems can be overcome by using different sequencing chemistry.

 

Check out the ABI sequencing chemistry handbook for more information.

-bob1-

We have sequenced several GC rich amplicons (those that had to be amplified with DMSO in the first place) and the ABI BigDye chemistry doesn't have problems with them with the standard protocol. But probably the smaller amplicons (300-500) behave differently than longer gDNA strands unless there is really tight hairpin formation or such.

 

In cases where the GC content would interfere with the cycle sequencing they may add some aditives like DMSO to the cycle sequencing as well. ABI also sells sequencing kits specificaly for GC rich sequences, where I suppose they already have aditives optimized for this. 

-Trof-

one can also use betaine when sequencing gc rich dna.

-mdfenko-