Duplex formation (hybridization positive)
No duplex formation (hybridization negative)
Unknown organism identified or detected
Figure 8-1 Principles of nucleic acid hybridization. Identification of unknown organism is established by positive hybridization (i.e., duplex formation) between a nucleic add strand from the known organism (i.e., the probe), and a target nudeic acid strand from the organism to be identified. Failure to hybridize indicates lack of homology between probe and target nucleic acid.
depending on the specific design of the hybridization assay.
Hybridization Steps and Components
Production and Labeling of Probe Nucleic Acid In keeping with the requirement of complementation for hybridization, the probe design (i.e., probe length and its sequence of nucleic acid bases) depends on the sequence of the intended target nucleic acid. Therefore, selection and design of a probe depends on the intended use, For example, if a probe is to be used for recognizing only gram-positive bacteria, the probe's nucleic acid sequence needs to be specifically complementary to a nucleic acid sequence common only to gram-positive bacteria and not to gram-negative bacteria. Even more specific probes can be designed to identify a particular bacterial genus or species, a virulence, or an antibiotic resistance gene that may only be present in certain strains within a given species.
In the past, probes were produced through a labor-intensive process that involved recombinant DNA and cloning techniques with the piece of nucleic acid of interest. More recently, probes are chemically synthesized using instrumentation, a service that is widely available commercially. This greatly facilitates probe development because the user need only supply the manufacturer with the nucleotide base sequence of the desired probe. The base sequence of potential
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