PCR problems:inverted repeats
horton at cis.umn.edu
Thu Jan 19 16:08:09 EST 1995
HARDIES at THORIN.UTHSCSA.EDU wrote:
: 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. <snip, snip>
: ...If the primers
: are within the inverted repeat, move them outside of it.
: You may be happier changing the strategy to two PCR products <snip>
Yeah, do that. But if that is just too easy, I wonder if it would help
to do REAL FAST cycling, like they do with the air cyclers and
capillary tubes. Presumably, with high primer concentrations, you could
get the primers to bind before the template snaps back into a hairpin.
Then, if you cycled fast enough, it might amplify... Or maybe not (the
effective local concentration of the complementary sequence in a hairpin
is really high. Might depend on how far apart the inverted repeats are.)
: 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.
Maybe it worked a little bit in each round? Slow cooling of the product
after the final denaturation should favor formation of the
(thermodynamically more stable) duplexes. Of course, this is all
Thermodynamics According to Bob.
This is an interesting problem - let us know how you solve it!
Bob Horton (Ph.D.!) /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS || with nine women pregnant you get a baby a month"
1479 Gortner Ave. /||\ -Werner von Braun. Disclaimer:"Bob who?"
St. Paul, MN 55108 ^^ horton at molbio.cbs.umn.edu/(612) 624-3790
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