Heteroduplex Analysis

[Song-Muh Jong]

Hi, 
      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.
     
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Song-Muh Jong                     TEL: (904) 392-0011
   BITNET:   sjj at ufbiot           FAX: (904) 392-6249
   Internet: sjj at icbr.ifas.ufl.edu