Agarose gel migration artifacts of PCR products.

Dennis J. Templeton djt2 at po.CWRU.Edu
Thu Dec 10 17:39:50 EST 1992


In a previous article, bsh at MED.PITT.EDU (Basavaraju Shankarappa) says:

>
>Hi Netfolks:
>	We have been trying to resolve the amplified product on a
>agarose gel.  Usually we have no problem.  But in one case we tried
>primers that contained lot of degeneracies.  When we run this product
>on an agarose gel, there is a kind of smearing effect in the lanes.
>This smear is clearly not caused by the presence of products of varied
>sizes.  The lane looks like the DNA appears to have aggregated and
>travels as a streak along the two edges of the lane.  We can also 
>faintly see the band at the appropriate positions.
>	We tried running the amplified product after diluting it and 
>also added SDS.  But it does not seem to go away.  Has anybody else
>seen this happen or has worked out a solution.  If I get a reply
>that solves the problem, I will post on the net.
>	Thanks 
>Raj Shankarappa
>Univ of Pittsburgh
>bsh at med.pitt.edu
>

Raj;

I once did a reaction of RNA's that amplified two species, one having an
insert of 50 bp not found in the other.  Instead of getting two bands, I
got 3, the extra one being slower migrating.  After a lot of work, I proved
that the "large" band is a hybrid between the two, and that it runs larger
because it has a loop sticking out.

I'll bet that with your degeneracies you havespecies that are hybrids
between two not-perfectly-complementary species, that migrate slowly and
smear because they are not true linears.

What to do?

Maybe run fewer cycles so that almost all of the products will result from
annealing of a primer and extention to a perfect DS linear.  After lots of
cycles most of the product results from reannealing of strands synthesized
in earlier cycles.  This helped in the situation I described above.

Maybe try reannealing the strands more stringently out of the reaction you
do now.  i.e., melt the strands at 95d and do a slow ramp down to a fully
annealed temp.  That way those strands most-perfectly complementary will
anneal at higher temperatures.  I'd try a slow ramp from maybe 85 to 60d in
PCR buffer.

let us know what helps; many of us are doing things with degeneracies now.

dennis



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