UVP HybriCycler - Manual (Page 8)

HC-3000 HybriCycler Hybridization Oven 8
Hybridization Techniques
There are really two main steps to a hybridization reaction: hybridizing two strands of
complimentary DNA and detection of the hybridized DNA. Nucleic acid hybridization is a
mechanism where strands of DNA in a single stranded state have their complements
bind together. The proximity of the DNA strands to each other determines the frequency
of the binding events and is in fact successful binding is proportional to their
concentration. The concentration of the target (nucleic acid you are looking for) is the
independent variable in all hybridization reactions.
Since the target concentration is usually the unknown variable, an excess of labeled
probe (what you use to find the target) will drive the reaction, thus decreasing the time
for the probe to hybridize to a target. This is simply increasing the chances of a probe
bumping into a target. But with an enormous amount of probe around (in solution or on
the surface of a membrane) the background signal will also be enormous. The typical
approach to correct for excess background (noise) on a membrane or slide hybridization:
wash in a low salt buffer as this favors the disassociation of unbound probe from the
membrane/slide and non-complementary DNA. In solutions a probe can be
enzymatically degraded by using a single strand specific nuclease.
Mechanisms of Nucleic Hybridization
Hybridization occurs with a process called
nucleation whereby the two separate
nucleic acid strands come into close
proximity of each other. A duplex region is
formed where a minimum of three bases of
one strand complements to those on the
second strand. If the remainder of the
strands are complementary, the two
strands will anneal or zipper together very
quickly. The rate limiting step in nucleic
acid hybridization is the duplex formation,
which again explains why probe to target
concentrations are critical.
Experimental Protocols
There are many different protocols available on the web, in journals, and in text
references and we reference several at the end of this text.