ligated pcr headache- ideas please

Euan R. Taylor etaylor at ccu.umanitoba.ca
Mon Mar 9 15:16:19 EST 1992


Is there anyone out there who has done ligated PCR?
I am trying to use this method to analyse the 5'end of a gene
using one primer to make a second strand from genomic DNA cut
to leave a 3' overhang, this leaves a blnt end (I'm using
sequenase ver1.0, isn't supposed to leave overhang). Them ligating on 
a linker which is blunt at one end and has an overhang at the other
so it wil only ligate in one orientation. 
Next I am using a second , specific internal primer and a primer from 
the linker sequence  as a pair to amplify the intervening DNA.
My substrate is genomic DNA cut with Pst 1 (gives 3' overhang so the
sequenase won't fil in). The fragment containing my target sequence is 
about 1.6kb and so should be suitable for PCR amplification even if I
am trying to get the whole fragment.
I am running controls which consist of th 5'end of the cDNA, again cut to give 
a 3' overhangand amplified the same way as the genomic samples. This works     
beautifully, starting from a few nanograms of material.
However even with the control working correctly in the adjacent tube, I dont   
seem to be getting my target sequence out of the genomic mix.
al the primer temperatures are high, and matched, lsequenase reaction, ligation
etc are obviously working to some reasonable extent.
I have tried partially  purifying the appropriate size fracton of the
digested genomic DNA on a gel before doing the reaactons, this reduces
non specific amplification, but I still dont get my target out.

I am running out of ideas and would appreciate any suggestions (preferably
helpful ones)
many tthanks
Euan Taylor

Etaylor at ccu.umanitoba.ca



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