ligating

Basavaraju Shankarappa bsh at MED.PITT.EDU
Thu Jul 29 19:55:46 EST 1993


>I want to attach double-stranded oligos to a mix of DNA fragments.  Then, I wan
>t to amplify these fragments by PCR using primers to the attached adapters.  To
> prevent insert chaining, I would like to phosphatase the fragments first, then
> ligate with the double stranded adapters.  The adapters can be synthesized wit
>h phosphorylated 5'nucleotides.  But the ligation, using T4, will only covalent
>ly attach the phosphorylated nucleotides in the adapter to one of the strands o
>f the fragment; the other strand, having been phosphatased, won't ligate.  I un
>derstand that when this is done in plasmids, the E. coli cloning bacterial enzy
>mes repair the unhooked strand.  But how can I do this for PCR?  Will Taq read
>or amplify through a break?  Would Klenow treatment do what is needed?  Please
>reply by email if you have tried this or know what will work.  Thanks.
> 
>Ken Weiss, PhD  Dept of Anthropology  Penn State University

I think "insert-chaining" should not be a serious one because you can make
a very good estimate of the number of cuts in the DNA and calculate the
mole ends and adjust it to the number of adapter molecules you include 
in the ligation.  As other alternatives,
	Two suggestions.
1. Include a restriction enzyme site at the end of the adapter and after 
ligating, digest with this enzyme to generate monomeric adapters at the
end of each DNA molecule.  

2.  If it is not very critical to amplify the entire mix of DNA fragments,
you can build a level of selection by incorporating a mixture of 3 bases
at the 3' end of the PCR primer.  The one base that is excluded will 
correspond to the one that will be formed by the multimer of adapters.

Regards,
Raj Shankarappa
bsh at med.pitt.edu
Univ of Pittsburgh



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