BIOSCI-REQUEST Mon Apr 17 10:16:58 1995
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>> To: genetic-linkage at net.bio.net>> From: t80bad1 at corn.cso.niu.edu (Brad A Didion)
>> Subject: locating ghost bands on RAPDs
>> Date: 17 Apr 1995 10:16:54 -0700
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>> In using RAPDs for detecting bands assoc. with trait loci, we've come
>> with a scenario in which the gel band is negatively assoc with the
trait of interest. In other words, when the band is missing (ghost) the
> is genotyped as positive because the trait of interest is assoc with
> ghost band. So, how can we identify the ghost band. Cloning/seq the
> negative band allowed PCR but this generated the neg band size in all
> animals...even those previously genotyped as positive (ghost band). My
> hope is that you're not confused as you ponder how to identify the
> positive allele. any help is appreciated. Brad.
I routinely analyze alleles corresponding to null (ghost) RAPDs in my
study of the genetics of Phytophthora infestans. Either PCR or
RFLP-based approaches are possible.
For the PCR approach, sequence the band that appears in repulsion to
your trait. Design longer primers (20-24 bp) and use these to amplify
bands from both genotypes. This succeeds about 3/4 of the time for me.
Presumably the null RAPD allele results from a small sequence difference
as compared to amplified band; using a longer primer (or a primer
binding to an internal part of the band) overcomes this difference and
allows amplification. Now you have amplified both "alleles" of the
locus. Next, you will need to distinguish between the alleles, since
usually the two alleles (bands) will be monomorphic. Several approaches
have worked for me:
1. Detect a restriction site difference between the bands (CAPS-PCR).
2. Sequence both bands and design allele-specific primers.
3. Detect differences by single-strand conformational polymorphism.
For the RFLP approach, use the RAPD band as a probe in Southerns to
detect RFLPs associated with your trait. Usually the RAPD band will
hybridize to the alternate alleles of the locus. Of course, sometimes
the alternate alleles will not be present at all in the other genotype.