Principles of nucleic acid hybridization

probe which typically consists of a homogeneous population of identified molecules (e.g. cloned DNA or chemically synthesized oligonucleotides) and a target which typically consists of a complex, heterogeneous population of nucleic acid molecules. If either the probe or the target is initially double-stranded, the individual strands must be separated, generally by heating or by alkaline treatment. After mixing single strands of probe with single strands of target, strands with complementary base sequences can be allowed to reassociate. Complementary probe strands can reanneal to form homoduplexes , as can complementary target DNA strands. However, it is the annealing of a probe DNA strand and a complementary target DNA strand to form a labeled probe-target heteroduplex that defines the usefulness of a nucleic acid hybridization assay. The rationale of the hybridization assay is to use the identified probe to query the target DNA by identifying fragments in the complex target which may be related in sequence to the probe (Figure 5.8 ).

• strand length - long homoduplexes contain a large number of hydrogen bonds and require more energy to separate them; because the labeling procedure typically results in short DNA probes, this effect is negligible above an original length (i.e. prior to labeling) of 500 bp;
• Figure 1.2 ), strands with a high % GC composition are more difficult to separate than those with a low % GC composition;
• chemical environment - the presence of monovalent cations (e.g. Na⁺ ions) stabilizes the duplex, whereas chemical denaturants such as formamide and urea destabilize the duplex by chemically disrupting the hydrogen bonds.

A useful measure of the stability of a nucleic acid duplex is the melting temperature (T _m ). This is the temperature corresponding to the midpoint in the observed transition from double-stranded to single-stranded form. Conveniently, this transition can be followed by measuring the optical density of the DNA. The bases of the nucleic acids absorb 260 nm ultraviolet (UV) light strongly. However, the adsorption by double-stranded DNA is considerably less than that of the free nucleotides. This difference, the so-called hypochromic effect, is due to interactions between the electron systems of adjacent bases, arising from the way in which adjacent bases are stacked in parallel in a double helix. If duplex DNA is gradually heated, therefore, there will be an increase in the light absorbed at 260 nm (the optical density₂₆₀ or OD₂₆₀ ) towards the value characteristic of the free bases. The temperature at which there is a midpoint in the optical density shift is then taken as the T _m (see Figure 5.9 ).

For mammalian genomes, with a base composition of about 40% GC, the DNA denatures with a T _m of about 87°C under approximately physiological conditions. The T _m of perfect hybrids formed by DNA, RNA or oligonucleotide probes can be determined according to the formulae in Table 5.2 . Often, hybridization conditions are chosen so as to promote heteroduplex formation and the hybridization temperature is often as much as 25°C below the T _m . However, after the hybridization and removal of excess probe, hybridization washes may be conducted under more stringent conditions so as to disrupt all duplexes other than those between very closely related sequences. Probe-target heteroduplexes are most stable thermodynamically when the region of duplex formation contains perfect base matching. Mismatches between the two strands of a heteroduplex reduce the T _m : for normal DNA probes, each 1% of mismatching reduces the T _m by approximately 1°C. Although probe-target heteroduplexes are usually not as stable as reannealed probe homoduplexes, a considerable degree of mismatching can be tolerated if the overall region of base complementarity is long (>100 bp; see Figure 5.10 ).

Increasing the concentration of NaCl and reducing the temperature reduces the hybridization stringency , and enhances the stability of mismatched heteroduplexes. This means that comparatively diverged members of a multigene family or other repetitive DNA family can be identified by hybridization using a specific family member as a probe. Additionally, a gene sequence from one species can be used as a probe to identify homologs in other comparatively diverged species, provided the sequence is reasonably conserved during evolution (see Figure 10.21 and Box 20.1 ).

Conditions can also be chosen to maximize hybridization stringency (e.g. lowering the concentration of NaCl and increasing the temperature), so as to encourage dissociation (denaturation) of mismatched heteroduplexes. If the region of base complementarity is small, as with oligonucleotide probes (typically 15 20 nucleotides), hybridization conditions can be chosen such that a single mismatch renders a heteroduplex unstable (see Section 5.3.1 ).

Reassociation kinetics is the term used to measure the speed at which complementary single-stranded molecules are able to find each other and form duplexes. It is determined by two major parameters: the starting concentration (C _o ) of the specific DNA sequence in moles of nucleotides per liter and the reaction time (t ) in seconds. Since the rate of reassociation is proportional to C _o and to t , the C _o t value (often loosely referred to as the cot value) is a useful measure. The C _o t value will also vary depending on the temperature of reassociation and the concentration of monovalent cations. As a result, it is usual to use fixed reference values: a reassociation temperature of 65°C and a Na⁺ concentration of 0.3 M NaCl.

Most hybridization assays use an excess of target nucleic acid over probe in order to encourage probe- target formation. This is so because the probe is usually homogenous , often consisting of a single type of cloned DNA molecule or RNA molecule, but the target nucleic acid is typically heterogeneous, comprising for example genomic DNA or total cellular RNA. In the latter case the concentration of any one sequence may be very low, thereby causing the rate of reassociation to be slow. For example, if a Southern blot uses a cloned b-globin gene as a probe to identify complementary sequences in human genomic DNA, the latter will be present in very low concentration (the b-globin gene is an example of a single copy sequence and in this case represents only 0.00005% of human genomic DNA). It is therefore necessary to use several micrograms of target DNA to drive the reaction. By contrast, certain other sequences are highly repeated in genomic DNA (see Section 7.3 ), and this greatly elevated DNA concentration results in a comparatively rapid reassociation time.

Because the amount of target nucleic acid bound by a probe depends on the copy number of the recognized sequence, hybridization signal intensity is proportional to the copy number of the recognized sequence. Single copy genes give weak hybridization signals, highly repetitive DNA sequences give very strong signals. If a particular probe is heterogeneous and contains a low copy sequence of interest, such as a specific gene, mixed with a highly abundant DNA repeat, the weak hybridization signal obtained with the former will be completely masked by the strong repetitive DNA hybridization signal. This effect can, however, be overcome by competition hybridization (see Box 5.3 ).

Box 5.4 ).