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Positive hybridization detected by visualization of color

Hybridized duplex labeled with acridinium

Positive hybridization detected by capturing emission of light

Figure 8-2 Reporter molecule labeling of nucleic acid probes and principles of hybridization detection. Use of probes labeled with a radioactive reporter, with hybridization detected by autoradiography (A); probes labeled with biotin-avidin reporter, with hybridization detected by a colorimetric assay (B); probes labeled with chemiluminescent reporter (i.e., acridinium), with hybridization detected by a luminometer to detect emitted light (C).

target genes or gene fragments for probe design also is easily accessed using computer on-line services for gene sequence information (e.g., GENBANK, National Center for Biological Information). In short, the design and production of nucleic acid probes is now relatively easy. Although probes may be hundreds to thousands of bases long, oligonucleotide (i.e., 20 to 50

bases long) probes are usually sufficient for detection of most clinically relevant targets.

All hybridization tests must have a means to detect hybridization. This is accomplished with the use of a "reporter" molecule that chemically, forms a complex with the single-stranded probe DNA. Probes may be labeled with a variety of such molecules, but most commonly radioactive (e.g., 32P, i2SI, or 3SS), biotin-avidin, digoxigenin, or chemiluminescent labels are used {Figure 8-2).

With the use of radioactively labeled probes, hybridization is detected by the emission of radioactivity from the probe-target mixture (see Figure 8-2, A). Although this is a highly sensitive method for detecting hybridization, the difficulties of working with radioactivity in a clinical microbiology laboratory have limited the use of this type of label in the diagnostic setting.

Biotinylation is a nonradioactive alternative for labeling probes and involves the chemical incorporation of biotin into probe DNA. Biotin-labeled probe-target nucleic acid duplexes are detected using avidin, a biotin-binding protein that has been conjugated with an enzyme such as horseradish peroxidase. When a cbromogenic substrate is added, the peroxidase produces a colored product that can be detected visually or £pectrophotometrically (see Figure 8-2, B) -

Other nonradioactive labels are based on principles similar to biotinylation. For example, with digoxigenin-labeled probes, hybridization is detected using anti-digoxigenin antibodies that have been conjugated with an enzyme. Successful duplex formation means the enzyme is present so that with the addition of a chromo-genic substrate color production is interpreted as positive hybridization. Alternatively, the antibody may be conjugated with fluorescent dyes that can be directly detected without the need for an enzymatic reaction tp produce a colored or fluorescent end product.

Chemiluminescent reporter molecules can be directly chemically linked to the nucleic add probe without using a conjugated antibody. These molecules (e.g., aaidinium) emit light so hybridization between a chemiluminescent-labeled probe and target nucleic add can be detected using a luminometer (see Figure S-2, Q. The chemiluminescent approach is used in one commercially available hybridization system (Gen-Probe, San Diego, Calif).

Preparation of Target Nucleic Acid. Because hybridization is driven by complementary binding of homologous nudeic add sequences between probe and target target nudeic add must be single-stranded and its base sequence integrity maintained. Failure to meet these requirements will result in negative hybridization reactions that are due to factors other than the absence of microbial target nudeic add (i.e., false-negative results).

Because the relatively rigorous procedures for releasing nudeic add from the target microorganism can be deleterious to the molecule's structure, obtaining target nudeic add and maintaining its appropriate conformation and sequence can be difficult. The steps in target preparation vary depending on the organism source of the nudeic add and the nature of the environment from which the target organism is being prepared (i.e., laboratory culture media; fresh dinical material, such as fluid, tissue, and stool; and fixed or preserved clinical material). Generally, target preparation steps involve enzymatic and/or chemical destruction of the microbial envelope to release target nucleic add, stabilization of target nucleic add to preserve structural integrity, and, if the target is DNA, dena-turation to a single strand, which is necessary for binding to complementary probe nucleic add.

Mixture and Hybridization of Target and Probe.

Designs for mixing target and probe nudeic adds are discussed later, but some general concepts regarding the hybridization reaction require consideration.

The ability of the probe to bind the correct target depends on the extent of base sequence homology between the two nudeic add strands and the environment in which probe and target are brought together. Environmental conditions set the stringency for a hybridization reaction, and the degree of stringency can determine the outcome of the reaction. Hybridization stringency is most affected by:

  • Salt concentration in the hybridization buffer (stringency increases as salt concentration decreases)
  • Temperature (stringency increases as temperature increases)
  • Concentration of destabilizing agents (stringency increases with increasing concentrations of formamide or urea)

With greater stringency, a higher degree of base-pair complementarity is required between probe and target to obtain successful hybridization (i.e., less tolerance for deviations in base sequence). Under less stringent conditions, strands with less base-pair complementarity (i.e./ strands having a higher number of mismatched base pairs within the sequence) may still hybridize. Therefore, as stringency increases, the spe-dfidty of hybridization increases and as stringency decreases, specificity decreases. For example, under high stringency a probe specific for a target sequence in Streptococcus pneumoniae may only bind to target prepared from this spedes (high spedfidty), but under low stringency the same probe may bind to targets from various streptococcal spedes (lower spedfidty). Therefore, to ensure accuracy in hybridization, reaction conditions must be carefully controlled.

Detection of Hybridization. The method of detecting hybridization depends on the reporter molecule used for labeling probe nudeic acid and on the hybridization format (see Figure 8-2). Detection of hybridization using radioactively labeled probes is done by exposing the reaction mixture to radiograph film (i.e., autoradiography). Hybridization with non-radioactively labeled probes is detected using co-lorimetry, fluorescence, or chemiluminescence, and detection can be somewhat automated using spectrophotometers, fluorometers, or luminometers, respectively. The more commonly used nonradioactive detection systems (e.g., digoxigenin, chemiluminescence) are able to detect approximately 104 target nucleic acid sequences per hybridization reaction.

Hybridization Formats

Hybridization reactions can be done using either a solution format or solid support format.

Solution Format. In the solution format, probe and target nucleotide strands are put together in a liquid reaction mixture that facilitates duplex formation, so hybridization occurs substantially faster than with the use of a solid support format. However, before detection of hybridization can be accomplished some method must be used to separate hybridized, labeled probe from nonhybridized, labeled probe (i.e., "background noise"). Separation methods include enzymatic (e.g., SI nuclease) digestion of single-stranded probe and precipitation of hybridized duplexes, hydroxyapatite or charged magnetic microparticles that preferentially bind duplexes, or chemical destruction of the reporter molecule (e.g., acridinium dye) that is attached to un-hybridized probe nucleic acid. After the duplexes have been "purified" from the reaction mixture and the background noise minimized, hybridization detection can proceed by the method appropriate for the type of reporter molecule used to label the probe (Figure 8-3).

Solid Support Format Either probe or target nucleic adds can form a complex to a solid support and still be capable of forming duplexes with complementary strands. Various solid support materials and common solid formats exist, induding filter hybridizations, south-em hybridizations, sandwich hybridizations, and in situ hybridizations.

Filter hybridizations have several variations. By one approach, the target sample, which can be previously purified DNA, the microorganism containing the target DNA, or the clinical specimen that contains the microorganism of interest, is affixed to a membrane (e.g., nitrocellulose or nylon fiber filters). Tb be able to identify specimens, samples are usually oriented on the membrane using a template or grid. The membrane is then processed to release target DNA from the microorganism and denature it to a single strand. A solution containing labeled probe nudeic add is used

A Probe and target nucleic acids mixed in solution

  • Reporter-labeled probe nucleic acid
  • Target nucleic acid

B Hybridization

  • Duplexes
  • Unbound, labeled probe

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