digestion of genomic DNA

Tom Knight tk at csail.mit.edu
Mon Oct 18 08:31:11 EST 2004

grace_tsoi2 at hotmail.com writes:
>   I am trying to digest the genomic DNA of my strain, but find that
>   it doesn't work with the enzyme Sau3A I after many hours.  My guess is
>   there are stuff along with the genomic DNA which prevents Sau3A I
>   from working.  My question is how and what exactly makes it fail
>   to work.

You don't say what your strain is, but there are many bacteria that
have methylases which add methyl groups selectively to A or C residues
in genomic DNA.  My organism, for example, methylates the C in the
sequence GATC, and is completely resistant to Sau3AI digestion, as
well as resistant to BamHI digestion, due to the internal GATC site in
the GGATCC recognition sequence for BamHI.  So although your DNA may
be inhibiting the reaction, it may also be that the GATC sites are
5-methyl C methylated.

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