Principles of nucleic acid hybridization
5.2.1. Nucleic acid hybridization is a method for identifying closely related nucleic acid molecules within two populations, a complex target population and a comparatively homogeneous probe population
Definition of nucleic acid hybridization
Nucleic acid hybridization involves mixing single strands of two sources of nucleic acids, a probe which typically consists of a homogeneous population ofidentified 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).
Melting temperature and hybridization stringency
Denaturation of double-stranded probe DNA is generally achieved by heating a solution of the labeled DNA to a temperature which disrupts the hydrogen bonds that hold the two complementary DNA strands together. The energy required to separate two perfectly complementary DNA strands is dependent on a number of factors, notably:
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;
base composition - because GC base pairs have one more hydrogen bond than AT base pairs (see 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 (Tm). 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 Tm (see Figure 5.9).
For mammalian genomes, with a base composition of about 40% GC, the DNA denatures with a Tm of about 87°C under approximately physiological conditions. The Tm 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 Tm. 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 Tm: for normal DNA probes, each 1% of mismatching reduces the Tm 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 thehybridization 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).
5.2.2. The kinetics of DNA reassociation are defined by the product of DNA concentration and time (Cot)
When double-stranded DNA is denatured, for example by heat, and the complementary single strands are then allowed to reassociate to form double-stranded DNA, the speed at which the complementary strands reassociate will depend on the starting concentration of the DNA. If there is a high concentration of the complementary DNA sequences, the time taken for any one single-stranded DNA molecule to find a complementary strand and form a duplex will be reduced. 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 (Co) 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 Co and to t, the Cot value (often loosely referred to as the cot value) is a useful measure. The Cot 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).
5.2.3. A wide variety of nucleic acid hybridization assays can be used
Early experiments in nucleic acid hybridization utilized solution hybridization, involving mixing of aqueous solutions of probe and target nucleic acids. However, the very low concentration of single copy sequences in complex genomes meant that reassociation times were inevitably slow. One widely used way of increasing the reassociation speed is to artificially increase the overall DNA concentration in aqueous solution by abstracting water molecules (e.g. by adding high concentrations of polyethylene glycol).
An alternative to solution hybridization which facilitated detection of reassociated molecules involved immobilizing the target DNA on a solid support, such as a membrane made of nitrocellulose or nylon, to both of which single-stranded DNA binds readily. Attachment of labeled probe to the immobilized target DNA can then be followed by removing the solution containing unbound probe DNA, extensive washing and drying in preparation for detection.
This is the basis of the standard nucleic acid hybridization assays currently in use. More recently, however, reverse hybridization assays have become more popular. In these cases, the probe population is unlabeled and fixed to the solid support, while the target nucleic acid is labeled and present in aqueous solution. Note, therefore, that probe and target are not primarily distinguished by which is the labeled and which is the unlabeled population. Instead, the important consideration is that the target DNA should be the complex imperfectly understood population which the probe (whose molecular identity is known) attempts to query. Depending on the nature and form of the probe and target, a very wide variety of nucleic acid hybridization assays can be devised (Box 5.4).
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