Help! A question about SSCP

Beth Wapelhorst weazel at animal.blarg.net
Wed Sep 9 12:31:30 EST 1998


What you need to do is cut out the individual SSCP bands out of hte
acrylamide gel and rePCR them and then sequence those PCR products. Mark
the original gel with some radioactive ink and reexpose it on film. Using
the marks, align the film and the gel to precisely cut out the gel bands.
Soak the cut out bands in 50 ul 10 mM Tris and use 5 ul in a new PCR
reaction. When you sequence these reactions, you will only be seeing one
allele instead of two, so heterozygosity won't hide the mutation.

Also, you should try MDE gel solution + 5% glycerol for SSCP gels instead
of acrylamide - your mutations will show up a lot easier.

beth


: > I am new to this newsgroup, please forgive me if my behavir is wrong
: > or
: > rude.
: > I am looking for a mutation site of  BRCA1 and BRCA2 gene, the
: > material
: > of DNA is mostly coming from paraffin.
: > I have done SSCP for hundreds of times ,some of them indicate the
: > mutation definitaly. However, the results of DNA sequence always beat
: > me:there is no mutation at all.
: > I know there must be someone who would tell me why.
: > Any suggestion will be highly appreciated.

: Overall, SSCP will only detect 50 to 70% of mutations. It's not a super
: easy technique ti use, but thoise researchers who do get it to work are
: fairly happy with the results. SSCP will not give you any information
: other than the fact that a mutation is there (or not), and sequencing is
: still the gold standard for locating and fully characterizing
: mutations.  There are other techniques out there, as well.


--
sugar beth wapelhorst              "I don't think I'm alone when I say I'd
weazel at blarg.net                    like to see more and more planets fall
www.blarg.net/~weazel               under the ruthless domination of our
                                    solar system." - Jack Handey



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