RAPDs and PCR

[Michael Gillings]

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

Michael


Dr Michael Gillings
Senior Research Scientist
Biological and Chemical Research Institute
PMB 10, Post Office Rydalmere
NSW 2116
AUSTRALIA

Ph   61-2-843-5709
Fax  61-2-630-4475
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)
> 
> 
>