Nucleic acid hybridization assays exploit the specificity of hybridization. A stable double-stranded hybrid (duplex) forms only when there is a significant amount of base pairing between the two sequences (which can be DNA, RNA, or oligonucleotide sequences). Because the stability of the duplex depends on the extent of base matching, assay conditions can be chosen to allow perfectly matched duplexes only or to allow degrees of base mismatching.
Hybridization assays can be carried out in many different ways, with multiple applications in both research and diagnostics. But there is a common underlying principle: a known, well-characterized population of nucleic acid molecules or synthetic oligo nucleotides (the probe population) is used to interrogate an imperfectly understood population of nucleic acids (the test sample). To do that, as required, both nucleic acid populations must be separated into single strands and then mixed so that single probe strands can form artificial duplexes with complementary strands in the test sample.
Because the object of a hybridization assay is to use the probe to identify complementary or partially complementary test-sample strands, the probe–test-sample duplexes need to be labeled in some way so that they can be identified. To do that, either the probe or the test sample needs to be labeled at the outset (see Figure 1 for one approach, where it is the probe that is labeled). As described in Box 6.2, different systems can be used to label nucleic acids and oligonucleotides, but in the former case the methods normally involve incorporating labeled nucleotides during DNA or RNA synthesis.
Fig1. Heteroduplex formation in a nucleic acid hybridization assay. For ease of illustration we consider here a homogeneous probe population, consisting of a single type of defined nucleic acid that is labeled (shown by red asterisks), and an unlabeled test sample made up of many different nucleic acids. For the assay to work any double-stranded molecules need to be denatured to give single strands. Thereafter, single-stranded probe nucleic acids are mixed with single-stranded test sample nucleic acids. Strands with complementary sequences are then allowed to anneal. Many of the fragments that had previously been base paired in the two populations will re-anneal to reconstitute original homoduplexes (bottom left and bottom right). In addition, new artificial duplexes will be formed between (usually) partially complementary probe and test sample sequences (bottom center). The hybridization conditions can be adjusted to favor formation of the novel duplexes. In this way, probes can selectively bind to and identify closely related nucleic acids within a complex nucleic acid population.
Using high- and low-hybridization stringency
A hybridization assay can be used to identify nucleic acid sequences that are distantly related from a given nucleic acid probe. We might want to start with a DNA clone from a human gene and use that to identify the corresponding mouse gene. The human and mouse genes might be significantly different in sequence, but if we choose a long DNA probe and reduce the stringency of hybridization, stable heteroduplexes can be allowed to form even though there might be significant base mismatches (Figure 2A).
Fig2. Using low- or high hybridization stringency to detect nucleic acid sequences that are distantly related or show perfect base matching with a given probe. In any hybridization assay we can control the degree of base matching between complementary strands in the probe and test sample. (A) If, for example, we increase salt concentrations and/or reduce the temperature, we lower hybridization stringency. In some circumstances a long probe strand can form a thermodynamically stable duplex with a comparable but distantly related strand within the test DNA (or RNA), even though there might be significant base mismatching. (B) Alternatively, we can use high temperatures and low salt concentrations to achieve high-hybridization stringency that might allow only perfect base matching. That is most easily achieved using a short oligonucleotide probe and it allows assays to discriminate between alleles that differ at a single nucleotide position. Labeling of nucleic acids and oligonucleotides is indicated by red asterisks.
Conversely, we can choose more stringent hybridization conditions to accept only perfect base matching. If we choose an oligonucleotide probe, we can use a high- hybridization stringency so that the only probe–test duplexes that can form are ones that contain exactly the same sequence as the probe (Figure 2B). That can happen because a single mismatch out of, say, 18 base pairs can make the duplex thermodynamically unstable. Oligonucleotides can therefore be used to identify alleles that differ by a single nucleotide (allele-specific oligonucleotides).
