Detection of Nucleic Acids by Hybridisation
Copyright 1992, 1998
Hybridisation is a term used to describe the specific complementary association due to hydrogen bonding, under experimental conditions, of single-stranded nucleic acids. It should more properly be referred to as "annealing", as this is the physical process responsible for the association: two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel "hybrid" helical molecule. One may make ones nucleic acid single-stranded for the purpose of annealing - if it is not single-stranded already, like most RNA viruses - by heating it in 0.01M NaCl to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling to +0oC: this ensures the "denatured" or separated strands do not re-anneal.
Alternatively, one may denature DNA reversibly by treatment with 0.5M NaOH: this does not work for RNA, as this hydrolyses under these conditions.
The answer is simple: nucleic acid hybridisation on membrane filters is a simple, sensitive, and specific means of detecting nucleic acid sequences of interest. One immobilises "target" nucleic acid - denatured so as to be effectively single-stranded - on an absorptive, porous membrane, and then anneals to it an appropriately "tagged" or "labelled" single-stranded probe nucleic acid. After washing off unannealed probe, one detects the immobilised hybrid by means of the label: this is often 32P incorporated into a nucleotide, which allows autoradiographic or scintillometric detection.
One may also use non-radioactive labels and detection systems, for sensitivities of detection down to picogram levels. The system of choice at the moment appears to be the Boehringer Mannheim DIG (digoxigenin) non-radioactive labelling and detection kit, which uses digoxigenin-11-dUTP as a substituted nucleotide which is enzymatically incorporated into DNA.
The mechanism of immobilisation of nucleic acids on membranes is not fully understood: nitrocellulose strongly binds only ss-nucleic acids (ssNA), under conditions of high salt (>1M NaCl), and has to be heated at 80oC in a vacuum to irreversibly attach the NA; nylon membranes (Hybond-N, GeneScreen) bind all nucleic acids under a wide range of salt concentrations, and irreversible or covalent attachment can be achieved by UV irradiation for 5 min or less, or by treatment with 0.4M NaOH.
The complementary association of two strands of polynucleotides
is the basis for replication of all organisms; the complexity inherent in the sequence of the molecules renders the association extremely specific for any molecule longer than sixteen nucleotides. This is easily understood if one considers the combinatorial possibilities of given lengths of "probe" sequence: there is a ¼ chance (4-1) of finding an A, G, C or T (U for RNA) in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every
416 bases (=4 294 967 296, or 4 billion):
this is about the size of the human genome, and 1000x greater than the genome size of E. coli.
Thus, the association of two nucleic acid molecules - presumed to be at least a few hundred bases long - is an extremely sequence-specific process, far more so than the widely-used specificity of monoclonal antibodies in binding to specific antigenic determinants. The correct annealing of two sequences to each other does, however, depend on the physical and chemical solution conditions under which the reaction takes place.
For example, all double-stranded nucleic acids - whether dsDNA, dsRNA or RNA:DNA hybrids - have specific "melting temperatures", which depend mainly upon their specific guanine+cytosine content, but also upon whether they are DNA, RNA, or a mixture (RNA:RNA hybrids have the highest melting temperatures, followed by DNA:RNA hybrids, then dsDNA), and upon the ionic strength of solution.
The melting temperature is also dependent upon the length of the sequences to be annealed: the shorter the probe sequence, the lower the melting temperature. The degree of sequence mismatch also determines the effective melting temperature of a hybrid: Tm decreases by about 1oC for every 1% of mismatched base pairs. It therefore makes sense to maximise probe length in order to minimise Tm reduction due both to length and degree of sequence mismatch. Under standard conditions of annealing (0.8M NaCl, neutral pH) one may calculate the melting temperature ™ of any given DNA hybrid as shown:
Tm = 81.5oC + 0.41(%G + %C) - 550/n
where n=probe length (no. nucleotides).
One can see that the reduction in Tm becomes negligible for probes of length 200 nt or greater. Thus, one may vary the specificity of association of a specific single-stranded "probe" and a target by varying the incubation temperature of the annealing reaction: the higher the temperature, the higher the specificity of the reaction - and the lower the likelihood of annealing taking place.
The successful use of nucleic acids as probes for sequences of interest therefore depends upon certain reaction conditions which are in turn determined by the physical properties (ie. length and sequence) of the probe. This leads to the concept of stringency of hybridisation: one increases the stringency by lessening the likelihood of non-homologous annealing. This can be done by simply increasing the temperature of incubation - bearing in mind that rate of hybridisation/annealing is maximal at about Tm - 25oC, and too high a temperature results in very slow annealing. An acceptable compromise is to anneal at a standard temperature (eg. 65oC), and then wash the annealed and immobilised hybrid molecules to varying degrees of stringency: the extent to which one should wash can be assessed by repeated autoradiography, if the probe is 32P-labelled, or by repeated colour assay of replicates in the case of non-radioactively labelled probe. Washing stringency may be increased by varying the ionic strength (from 1.0M NaCl to 0.02M), or varying the temperature (ambient to 65oC). One may also include SDS or other detergent in wash and in hybridisation buffers in order to decrease non-specific attachment of probe to the adsorptive membrane. For this reason a blocking or prehybridisation buffer is normally used before and during the annealing reaction, to block adsorptive sites on the membrane not occupied by target nucleic acid. This normally consists of buffer salts, detergent, protein, inert polymer material, and DNA.
It is possible to include various other constituents in annealing buffers, designed to increase the hybridisation rate, or the stringency, or both. Formamide is a helix destabiliser, and enables one to decrease annealing temperature: the presence of formamide decreases the Tm as shown:
TFm = Tm - 0.61(%formamide, w/v)
It is most often used in annealing reactions using RNA as target or probe, and especially with dsRNA hybrids, as these have high Tms which necessitate elevated reaction temperatures. Standard conditions using formamide would be 42oC with 50% formamide content in the annealing buffer. Formamide also decreases the rate of annealing, so one normally includes substances like dextran sulphate - a polyanionic polymer - as "molecular exclusion agents" to decrease the volume of solvent available to the probe. Polyethylene glycol is a far cheaper and equally effective substitute for increasing reaction rate. Too high a concentration of DS or PEG raises "background" or non-specific probe attachment to unacceptably high levels. Their effectiveness is also directly proportional to probe length, and they are useless when oligonucleotides of less than 50 nt in length are used as probes.
A standard hybridisation reaction, then, consists of probing an immobilised target sequence on a membrane with a labelled specific probe sequence : this is done by annealing the probe to the target under (usually) standard "hybridisation conditions" of 0.9M NaCl, 65oC, for 4-16 hr. Probes are usually molecules of DNA or cDNA, a few hundred nt to several kilobases long, cloned into and grown up as recombinant plasmids in E. coli, and purified by caesium chloride gradient centrifugation. One may also use nucleic acid directly purified from the organism of interest, but this is only really effective if this is a virus or a plasmid, as otherwise the probe length is too great, and the repeat number is too small to give appreciable signal. In other words, probes should not be too long, as otherwise one needs very high concentrations of nucleic acid in order to guarantee a sufficient number of copies of the sequence in order to give a detectable "signal" for detection purposes.