3 & 5' Overhangs...

[Andrew Healey]

David L. Haviland, Ph.D. (HAVILAND at KIDS.WUSTL.EDU) wrote:
: Hi fellow netters:


:                5'
: 		---------..
: 	    -------------..  3' overhang left by SacI
:            3'


: and 	5'
: 	----------..
: 	    ------..        5' overhang left by NheI
:             3'

: I'd like to be able to join these two ends and retain at the very least the 
: SacI site.  As I interpret the details of T4 Polymerase, T4 will only end
: fill the NheI site in the presence of dNTPs.  WITHOUT dNTPs T4 will remove
: the 3' overhang of the SacI site. 

And WITH the dNTP's present.


: Using T4 should, if I understand things correctly, give back the NheI site 
: at the expense of the SacI site.  Correct?  Unless I'm missing something T4
: can *not* end-fill the SacI site?  (It can't go 3'->5')

Yep, you'll get ---GCTAG     and      C---
                ---CGATC              G---

                  NheI                SacI

....which restores the NheI site only.

: I'd like to be able to keep BOTH sites but now I'm beginning to wonder if 
: that can even be done without the aid of a SacI-NheI linker (most likely 
: home made!).

Whatever the reason you need Sac I there, you can't do it with end filling.
T4 RNA ligase might do it with the unfilled ends juxtaposing, then do a
nick repair with T4 DNA ligase (I haven't done it...just a suggestion) or
alternatively make a CTAGAGCT phosphorylated oligo to stick the ends together
before ligasing.

Andrew
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