UVP HB-500 Minidizer - Manual (Page 10)

HB-500 Minidizer Hybridization Oven 10
Hybridization Techniques
There are really two main steps to a hybridization reaction: hybridizing two strands of complementary 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, in fact, successful binding is proportional to their concentration. The
concentration of the target (nucleic acid) is the independent variable in all hybridization reactions.
Since the target concentration is usually the unknown variable, an excess of labeled probes (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. However, with an enormous amount of probe around (in the 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 is to wash it 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.
1. Concentration of Species
Target: How much target molecule depends on the species you expect to find. Cellular
constituents may be expressed in large or small amounts; the trick is to start with enough target
(~25
µg) and determine experimentally.
Probe: Plan to have more probe than expected target. To answer questions about adding too
much probe, run an extinction experiment: serially increase the amount of target by a factor of two
and use a fixed amount of probe. Hybridize for a short length of time and quantitate the amount of
probe that has hybridized. As long as the signal increases and shows linearity there is excess
probe (Fig 2). If the signal levels off and a loss of linearity noticed, then the probe is not in excess
(Fig 2).
2. Length of Probe
The goal is to increase hybridization efficiency while minimizing background. In most cases probes
range from 20 – 1000 bps.