Hoefer HB1000 - Instrukcja (Strona 20)

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p11
Hybridization techniques
There are two main steps to a hybridization
reaction: 1) hybridizing two strands of DNA
carrying complementary sequences, and 2) detec-
tion of the hybridized DNA. Proximity of the
DNA strands determines the frequency of the
binding event and, therefore, success is depen-
dent on the DNA concentrations.
Since the concentration of the target nucleic
acid is usually unknown, it is an excess of labeled
probe DNA that drives forward the hybridization
reaction. This is simply increasing the chances
of a probe encountering a target. But with an
enormous amount of probe present (in solution
or on the surface of a membrane) the back-
ground signal will also be enormous. The typical
approach to correct for excess background on
a membrane or slide hybridization is to wash in
a low salt buffer as this favors the dissociation
of unbound probe from the membrane/slide and
from non-complementary DNA. In solutions, a
probe can be enzymatically degraded by using a
single-strand specific nuclease.