T4 DNA Ligase: Oligo to Sticky End

Keith Rand Keith.Rand at molsci.csiro.au
Thu Nov 27 18:41:55 EST 1997


In article <65e15m$leb$1 at nargun.cc.uq.edu.au>, Paul R. Rohde
<P.Rohde at mailbox.uq.edu.au> wrote:

> T4 DNA Ligase joins blunt ended or cohesive-ended double stranded
> molecules but does not join single stranded molecules.
> 
> My question:  Will T4 DNA Ligase join to a four base sticky end,a
> complementary oligonucleotide?  Yes, the oligo is single stranded,
> but the actual ligation site (or nick) is actually double stranded.
> This should work shouldn't it?  The oligo has been kinased to the 5'
> end.
> 
> 
> 
>                          5'-gatcnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn...
>      3'-NNNNNNNNNNNNNNNNNNNNCTAGnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn...
>                                ^^
>                              "nick"
> 
> 
>         (Oligo Capitals, ds Sticky End DNA lower case)

Suggest check the ref : Biotechniques 23  418-420  Zhao & Hortsch 1997

Describes how e.g the oligo CTAGGTAC can be used to join Xba1 end to a
KpnI end. Presumably the first ligation event is similar to that suggested
in your question.

-- 
Keith Rand,  Sydney Australia



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