1. It is important to understand the principals of molecular hybridization in order to be able to answer such questions as:
2. Hybridization kinetics: hybridization involves two steps: initial contact between complementary stretches of DNA strands and "zipping up" of the two strands to form a complete hybrid molecule. The first step is rate-limiting. The second proceeds very rapidly. Initial contacts occur as two complementary strands bump into each other as each is diffusing through solution (solution hybridization) or as one of them is diffusing through solution (membrane hybridization). Solution hybridization is bimolecular, and the rate of formation of hybrid molecules is proportional to the square of the concentration of single-stranded species (C):
dC/dt = -kC2
The kinetics of membrane hybridization are complex and depend upon the ratios of amounts of the membrane-bound nucleic acid and the probe. If the probe is in vast excess, the reaction is unimolecular, and the rate of hybridization is proportional to simple probe concentration. The rate of disappearance of the probe also depends on whether or not the probe is double-stranded or single-stranded, because the two strands of double-stranded probes will reanneal with each other (in solution, following bimolecular kinetics), thus becoming unavailable to hybridize with the target sequence on the membrane. If you wish to know the kinetics of hybridization with membrane-bound nucleic acid, it is best to determine the kinetics empirically, by experiment, rather than attempting to predict them theoretically.
3. Optimum hybridization conditions are determined by the melting properties of the DNA being studied (Tm) and by the ionic strength of the solution. Optimum hybridization rate is usually found 20-30° C below the Tm (0.77 M Na+=5 X SSC).
4. Tm is reduced about 1°C for each % mismatch.
5. The wash conditions provided in standard protocols are intended to maximize stringency (to prevent the detection of signals from DNA strands not perfectly complementary to the probe). If stringency is not a problem, signal can be increased by reducing wash temperature or by raising wash ionic strength.
6. General background during hybridization (signals from the entire membrane, not just from the section of the membrane containing bound DNA) cannot be reduced, relative to true signal, by increasing wash stringency. Increased low stringency washes frequently help. Do not use increased high-stringency washes or you will reduce your signal as fast as, or faster than, you reduce background! The most common causes of high background are (i) inadequate blocking of DNA-reactive sites on the membrane during pre-hybridization, (ii) partial drying of the membrane during hybridization, and (iii) trapping of unhybridized probe in the thin fluid layer between overlapping membranes.
This page was updated August 30, 1998.
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