oligo Southerns...HELP!

kmorris1 at opal.tufts.edu kmorris1 at opal.tufts.edu
Thu Jun 16 13:43:22 EST 1994

I hope someone will be able to help me... I'm going crazy with this problem!
I am trying to set up a competitive PCR to detect mutant and wild-type alleles
of p53 in a particular cell line.  I have made both WT and M competitor
constructs, and the PCR itself seems to be working well.  Both alleles from
genomic DNA, however, amplify the same size fragment so I have to use
differential hybridization with specific oligonucleotides to quantitate.  This
is where the problem seems to be.  I have made two oligos, 19mers, that differ
by a single nucleotide- the point mutation present in the M allele.  The oligo
that is specific for WT p53 works like a dream...only binds to WT sequences,
not M sequences, etc.  The M p53 specific oligo doesn't.  It seems to bind with
equal affinity to both alleles and by the time I can get reasonable specificity 
I'm losing binding to the M alleles as well :-(   I have tried making a second
mutant oligo from the other strand, because someone here thought that might
help.  Nope.
	So basically I'm looking for help from anyone who has tried allele
specific hybridizations before.  How did you pick your oligos?  What sort of
conditions do you use for your hybridizations (I'm using 6XSSC/1%SDS for both
hyb and washing)?  Any tricks/tips I haven't learned yet?  This is the last set
of experiments I have to do before I can write up my stuff and graduate :-)
	Thanks in advance for any advice!

Kelly C. Thome               kmorris1 at opal.tufts.edu
Immunology Graduate Program
Tufts University             phone 617-956-6906
Boston, MA 02111               fax 617-956-0337

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