ligated pcr headache- ideas please

twomey at helix.nih.gov twomey at helix.nih.gov
Mon Mar 16 17:18:46 EST 1992


In article <1992Mar9.201619.24894 at ccu.umanitoba.ca> etaylor at ccu.umanitoba.ca
(Euan R. Taylor) writes:
>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
>
You seem to be describing what FORS et al. did in Nucleic Acids Research
18:2793-2799, 1990.  I followed their technique brainlessly and it worked
perfectly. Hope this helps.
Patrick Twomey
twomey at helix.nih.gov



More information about the Methods mailing list