There are many types of hybridization assay but they all fall into two broad classes, as listed below.
• Labeled probe–unlabeled test sample (left image in Figure 1). This has been the traditional type of hybridization. The probe is often a single type of cloned DNA, such as represented in Figure 2, and it is usually labeled by using a polymerase to synthesize complementary DNA or RNA strands in the presence of one or more labeled nucleotides.
• Unlabeled probe–labeled test sample (right image in Figure 1). This type of hybridization is sometimes called reverse hybridization because it is the reverse of the traditional approach. It has become popular since the development of microarrays, where unlabeled complex probe populations bound to a surface are used to interrogate a labeled test sample, as detailed below.
Fig1. Standard and reverse hybridization assays and the use of solid supports to capture labeled probe–test sample duplexes. In both assays the unlabeled nucleic acid (or oligonucleotide) population is bound to a solid support and denatured and is exposed to an aqueous solution of the labeled nucleic acid (or oligonucleotide) population that has also, as required, been denatured. Single-stranded molecules in the labeled population can hybridize to complementary sequences in the unlabeled population and so become bound to the solid support. Other labeled sequences that have not bound, or have bound nonspecifically at incorrect locations on the support, can be washed off. The bound labeled nucleic acids can then be studied and are sometimes retrieved by washing at higher temperatures to break the hydrogen bonds connecting them to their unlabeled partner strands on the supports. In the past, most hybridization assays used the labeled probe/immobilized test sample scheme shown on the left (see Table 6.3), but microarray hybridization assays use the labeled test sample/ immobilized probe scheme shown on the right.
Fig2. 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.
The point of using labeled nucleic acids in a hybridization assay is to allow probe–test sample heteroduplexes to be identified. But how can we distinguish between the label in these duplexes and the label in the original labeled probe or labeled test-sample DNA? The answer is to immobilize the unlabeled nucleic acid or oligonucleotide population on a solid support (often plastic, glass, or quartz) and expose it to an aqueous solution of the labeled nucleic acid population. When labeled nucleic acid strands hybridize to complementary sequences on the solid support, they will be physically bound to the support. However, labeled molecules that do not find a partner on the support (or that stick non specifically) can be washed off. That leaves behind the complementary partners that the assay is designed to find (see Figure 1). Hybridization assays are used for different purposes, as described later; see Table 1 for some examples.
Table1. POPULARLY USED HYBRIDIZATION ASSAYS
