sjj at srcros1.pathology.ufl.edu
Mon Mar 14 17:25:31 EST 1994
I have questions about using Heteroduplex Analyssi to identify
mutations in a gene. The expected size of my PCR product is ~200 bp. After
PCR, I boiled the samples for 5 min, then quick chill on ice for 2 min before
letting them sit at room temp for overnight. After resolving on an 8% PAGE,
a doublet at ~ 500 bp, in addition to the expected single band at ~ 200 bp,
were seen. However, the same patterns were seen in all my samples, both
normal and patient genomic DNAs. The latter makes it unlikely that the
mobility changes represent a mutation in the patient samples.
1. Is it possible that the mobility changes were caused by secondary
structure formation driven by the heating and reannealing procedure? (samples
without heating and reannealing showed only one band at 200 bp)
2. If secondary structure formation can result in a "false positive",
how does one get around this problem?
3. What do you think is a good explanation for my observation?
Thank you very much.
Song-Muh Jong TEL: (904) 392-0011
BITNET: sjj at ufbiot FAX: (904) 392-6249
Internet: sjj at icbr.ifas.ufl.edu
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