Mismatch specific nicking

Basavaraju Shankarappa bsh at MED.PITT.EDU
Tue Jun 8 14:16:30 EST 1993


Hi:
Last week I had asked a question regarding any possibilities
for cutting specifically at a nick, without chewing the ss or ds DNA.
This was necessary to devise a method to identify the presence and
possibly quatitate the proportion of mutant sequences in a given
population of amplified products.

Although my hypothesis of using DNA polymerase in the absence of dNTPs
does seem to hold any water, I have had some very good suggestions.

This one is a compliment to Promega in view of the slack it has been
taking recently regarding its taq.  Leslie (I wish I knew the last name)
from Promega was extremely helpful in that 
She managed to find a procedure that at least in theory can solve my
problem.  This procedure is based on oligonucleotide ligation assay
where you can use one primer with a biotin label and other with a 
digoxigenin label and allow them to ligate.  Two sets of primers can 
be made each of which specific for the mutant or the wild type.  
Using a avidin capture system followed by anti-dig antibodies should
enable identification and quantitation of the variants.
Automated DNA diagnostics using an ELISA based oligo ligation
assay.  Nickerson et al.,(Leroy Hood's lab) PNAS 87:8923.  As an
obvious extension of this procedure one can also use the ligation
chain reaction with appropriate modifications.  Ofcourse, as pointed 
out by Johan, you should know the exact location of the mutation.
(in our case we do).

Second procedure looks pretty good to me.  This was courtesy of
Johan de Boer (jdboer at sol.uvic.ca).  Here the heteroduplex with 
mismatches is treated with hydroxylamine and osmium tetroxide and
cleaved with piperidine to cleave at C and Ts.  I guess if there is
only one mismatch, it should be possible to detect it.  The ref is
Cotton et al., PNAS 85: 4397.  

If anybody has better ideas, "I am all ears".

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



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