RAPDs and PCR
gillinm at agric.nsw.gov.au
Wed Feb 21 23:50:34 EST 1996
I have heard of similar problems to this one, particularly when there is
internal homology within a RAPD product, or where there is homology
between two (or more) distinct RAPD fragments.
It is possible, for instance, that your RAPD fragment has multiple sites
that are partially homologous to the primer used: result, multiple products
from a single DNA species. It is also possible that the larger fragment is
itself an artefact; some sort of strange product dimer for instance.
I have two suggestions that might overcome your problem. When the RAPD
fragment is recovered from the gel, use a higher annealing temperature in
the second RAPD. You might go above the Tm, since you know that the RAPD
fragment has exact homology to the primer at either end.
Secondly, why not try hybridisation with the smaller RAPD fragment? ie
purify, amplify and label the low molecular wt product. It may well be
homologous to the larger fragment, and will certainly be easier to
recover from the agarose and hybridise with.
Best of luck
Dr Michael Gillings
Senior Research Scientist
Biological and Chemical Research Institute
PMB 10, Post Office Rydalmere
On 21 Feb 1996, Doug Johnson wrote:
> We are trying to use RAPD bands as S-blot probes. The idea is to gel
> extract and PCR using the same primer and 32-P. But when the band is
> extracted and re-amplified(same primer, same RAPD conditions are used),
> we observed lower MWt bands in addition to the original one.If the
> HMWt band is re-isolated from the second gel and amplified once
> again, we still see the LMWt bands. Has anyone seen this? Is this a
> problem with band to band separation when the agarose gel is used to
> prepare fragments? All suggestions are welcome and appreciated.
> Doug Johnson (djohnson at oreo.uottawa.ca)
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