amp/labelling with Klenow

Marc Crepeau via methods%40net.bio.net (by mcrepeau from ucdavis.edu)
Thu Nov 20 21:06:48 EST 2008


I'm looking at a NimbleGen protocol for DNA amplification/labelling
and I'm a little confused about how it works.  They start with 1 ug of
denatured DNA and add Cy-3- or Cy-5-labelled random 9mers, dNTPs and
Klenow fragment.  Then they incubate at 37C for two hours.  The result
is several micrograms of labelled DNA.  My question is how do you get
greater than two-fold amplification from this isothermal reaction?  I
figure there must be some strand displacement going on, and I know
Klenow has strand displacement activity, but doesn't the displacement
require a nick just downstream of the primer binding site?


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