Characteristics to Identify Prokaryotes

Increasingly, genotypic characteristics are being used to identify microorganisms, particularly those that are difficult to cultivate. DNA probes and polymerase chain reaction (PCR) can both be used to detect nucleotide sequences that are unique to a given organism. DNA sequencing has made identification possible of organisms that cannot be grown in culture. ■ nucleic acid probe, pp. 225,236 ■ polymerase chain reaction, pp. 229,239 ■ sequencing, pp. 229,237

Nucleic Acid Probes to Detect Specific Nucleotide Sequences

A nucleic acid probe can be used to locate a unique nucleotide sequence that characterizes a particular species of microorganism (figure 10.8). The probe is a single-stranded piece of nucleic acid, usually DNA, that has been labeled with a detectable tag, such as a radioisotope or a fluoresent dye. It is complementary to the sequence of interest.

Fluorescence in situ hybridization (FISH) is increasingly being used to observe and identify intact microorganisms in environmental samples and clinical samples. By using a probe that binds to certain ribosomal RNA (rRNA) sequences, either specific species or groups of related organisms can be identified; characteristics of rRNA that make it ideal for this purpose will be described shortly. ■ fluorescence in situ hybridization, p. 226

Methods that employ nucleic acid probes to detect DNA sequences in a specimen generally rely on some type of an amplification step. For example, in some cases the bacteria in the specimen must first be cultured on an agar plate so that each cell can multiply to form a colony containing well over a million cells. In other cases, a preliminary in vitro DNA amplification step is required.

Amplifying Specific DNA Sequences Using the Polymerase Chain Reaction

The polymerase chain reaction (PCR) can be used to amplify a specific nucleotide sequence present in nearly any environment (see figure 9.11). This includes DNA in samples such as body fluids, soil, food, and water. The technique can be used to detect organisms that are present in extremely small numbers as well as those that cannot be grown in culture.

To use PCR to detect a microbe of interest, a sample is first treated to release and denature the DNA. Specific primers and other ingredients are then added to the denatured DNA form-

Organism X DNA

Probe

Organism X DNA

Probe

Double-stranded DNA sequence unique to organism X.

DNA is labeled, then denatured to become a probe.

Double-stranded DNA sequence unique to organism X.

Unknown organism

(double-stranded DNA)

Denatured

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DNA is labeled, then denatured to become a probe.

Organism X probe is added to denatured, single-stranded DNA of unknown organism.

Single-stranded DNA

If probe does not bind to DNA, then unknown organism is not organism X.

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If probe binds to DNA, then unknown organism is organism X.

Figure 10.8 Nucleic Acid Probes to Detect Specific DNA Sequences The probe, which is a single-stranded piece of nucleic acid that has been labeled with a detectable marker, is used to locate a unique nucleotide sequence that identifies a particular known species of microorganism.

ing the components of the PCR reaction (see figure 9.23). Some information about the nucleotide sequence of the organism must be known in order to select the appropriate primers. After approximately 30 cycles of PCR, the DNA region flanked by the primers will have been amplified approximately a billion-fold (see figure 9.24). In most cases, this results in a sufficient quantity for the amplified fragment to be readily visible as a discrete band on an ethidium bromide-stained agarose gel. Alternatively, a DNA probe can be used to detect the amplified DNA. ■ ethidium bromide, p. 236 ■ gel electrophoresis, p. 236 ■ Taq polymerase, pp. 229,240

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