PCR problems:inverted repeats

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Mon Jan 16 16:31:28 EST 1995


Namrata Srivastava wrote:

>	Im trying to PCR a 3-4 Kb DNA fragment which contains 1.6 Kb of 
> inverted repeat sequence. I get a shorter product which would correspond 
> to a secondary DNA structure loop (DNA hybridizing to itself) hence the taq
> is unable to read through the entire template.

If you mean that there is a very long inverted repeat, then I'm not
confident that you can succeed.  If it's a series of small repeats,
then the procedures for amplifying through templates with secondary
structure might work because they increase the denaturing conditions
during the polymerization.  But if the stem that snaps back is as
stable as the primed duplex, then there's no option but to try to
synthesize with strand displacement.  I haven't seen any
characterization of the different enzymes capability to do this in the
literature, and I've always assumed that they can't; but you could try
some different enzymes to see if they might work.    If the primers
are within the inverted repeat, move them outside of it.  
    You may be happier changing the strategy to two PCR products, each
of which crosses just one of the repeat segments.  You'll still have
trouble getting the reaction started, but once started it'll behave
like ordinary PCR.  Or if you know some restriction sites to use, you
can cut the inverted repeat apart (I'm guessing a transposon-like
structure with a large segment between the inverted repeats).
    Your interpretation of the smaller band requirers that the
amplification actually worked, if I understand it.  I doublt this,
because the strands synthesized in the last cycle would still be
duplexed; so I don't understand how you would get all snap back and no
duplex.  I'd guess you probably rescued some rearranged product.

Hope this helps.

Steve Hardies, Dept. of biochemistry, Univ. of Texas HSC at San
Antonio
Hardies at uthscsa.edu





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