Restriction Enzyme selection for Hi-C

[chuanhe yu]

Dear all, 

I am trying to design a hi-C protocol in our lab. Previous the 3C analysis at our interested locus with enzyme Pst I works very well. The Hind III is not suitable for our locus analysis. 

I checked the hi-C protocol.  Most people use the enzyme which produces the 3’ overhang.   Have you ever used the 5’ overhang enzyme for hi-C?  Whether we can use the same enzyme (Pst I) to do Hi-C in which I can use T4 DNA polymerase to do biotin label at the 3’end? Thank you very much. 

Chuanhe

[Elena Stamenova]

Dear Chuanhe,

Restriction enzymes that leave 5’ recessed ends (3’ overhangs) such as PstI (CTGCA’G) are not suitable for biotin labeling with the Hi-C protocol we use. Only restriction enzymes that produce 5’ overhang can be used in Hi-C because only the 5’ overhang could be filled-in by DNA Polymerase I, Large (Klenow) fragment. Both T4 DNA Polymerase and Klenow fragment of DNA Polymerase I can remove the 3’ overhang formed by PstI through their 3’->5’ exonuclease activity.

I hope that this helps.

Best regards,

Elena

[chuanhe yu]

Thanks.

Chuanhe