[Biopython] Restriction enzymes and sticky ends

[Mark Budde]

Thanks Peter, that explains it. BsaI is indeed a weird enzyme, a TypeIIs
restriction enzyme. These enzymes cut a defined distance outside of their
recognition sequence. The utility of these enzymes is that by tagging the
cut sites on the end of your primers, you can generate whatever sticky ends
you desire. Furthermore, because it cuts outside of its recognition
sequence, you can incubate a number of these fragments together with both
restriction enzyme and ligase, and the fragments will assemble into the
final product without subcloning. This is because stciky ends are generated
without the corresponding recognition site, so their ligation is
irreversible. This is called GoldenGate cloning.
-Mark


On Mon, Apr 8, 2013 at 2:32 AM, Peter Cock <p.j.a.cock at googlemail.com>wrote:

> On Sun, Apr 7, 2013 at 9:15 PM, Mark Budde <markbudde at gmail.com> wrote:
> > Thanks for doing some digging on my behalf, Peter. After I posted my
> email
> > last night, I started looking through the Bio.Restriction code myself.
> You
> > response is helpful, I was having trouble seeing how the cut site was
> > encoded for each strand. I think Bjorn's python-dna might be a better
> > starting place for me than Bio.Restriction, as it already has some of the
> > functionality I was looking for.
>
> Fair enough.
>
> > However, to you question, I'm still not quite getting the cut sites. You
> > example with EcoRI makes complete sense, but I can't figure out the
> pattern
> > for some other enzymes, such as BsaI, which is why I got confused
> initially.
> > If you repeat that protocol for BsaI, the results don't match up.
> >
> > In [80]: BsaI.elucidate()
> > Out[80]: 'GGTCTCN^NNNN_N'
> >
> > In [81]: BsaI.fst5
> > Out[81]: 7
> >
> > In [82]: BsaI.fst3
> > Out[82]: 5
> >
> > In [83]: BsaI.site
> > Out[83]: 'GGTCTC'
> >
> > Based on this, I would expect that BsaI.fst3 should yield
> > "11" but it yields 5.
>
> I think you are counting from the wrong reference point.
> Using Python style indexing would only allow cleavage
> points within the recognition site to be described.
>
> BsaI is a weird enzyme, and appears to be handled by the
> Ambiguous class in Bio/Restriction/Restriction.py - which
> says this is for enzymes for which the overhang is variable.
>
> >>> from Bio.Restriction import Bsal
> >>> BsaI.is_ambiguous()
> True
> >>> BsaI.is_defined() # is there a consistent site?
> False
> >>> BsaI.is_unknown()
> False
> >>> BsaI.fst5
> 7
> >>> BsaI.fst3
> 5
> >>> BsaI.elucidate()
> 'GGTCTCN^NNNN_N'
>
> This subclass has a more complicated elucidate method,
> but gives the same string as the REBASE website, so this
> is deliberate: http://rebase.neb.com/rebase/enz/BsaI.html
>
> The 5' cut site of 7 clearly means this is downstream of
> the 6 bp recognition site. This appears to be counted
> from the start (left) of the restriction site.
>
> From the illustration the 3' cut side is also to the right of
> the 5bp recognition site. It appears the number is counted
> from the end (right) of the recognition site, where positive
> as in BsaI means to the right (after the recognition site)
> while negative as in EcoRI means to the left (within the
> recognition site).
>
> Peter
>
> P.S. Please remember to CC the mailing list, e.g. reply all.
> Unless people say explicitly that they have done this deliberately,
> I generally assume taking a public discussion off list is accidental.
>