cloning ghost (null) RAPD bands

howard judelson judelson at UCRAC1.UCR.EDU
Mon Apr 24 19:53:13 EST 1995


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> From: t80bad1 at corn.cso.niu.edu (Brad A Didion)
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> Subject: locating ghost bands on RAPDs
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> 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 
up
> 
> 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 
animal
> is genotyped as positive because the trait of interest is assoc with 
the
> 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.






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