Nvh

Probe is added

Probe hybridizes to complementary sequences on membrane

Membrane is washed; autoradiograph detects hybridized probe

Probe is added

Probe hybridizes to complementary sequences on membrane

Autoradiograph -

Autoradiograph -

Membrane is washed; autoradiograph detects hybridized probe

Match dark spots with original plate

Figure 9.7 The Steps of a Colony Blot This technique is used to determine which colonies on a plate contain a given DNA sequence. In this example, the probe is labeled with a radioactive isotope, which can be detected using autoradiography.

using a fluorescence microscope. To examine prokary-otes, a probe that binds to ribosomal RNA (rRNA) is generally used. Because actively growing cells can have thousands of copies of rRNA, this increases the sensitivity of the technique. Other characteristics of rRNA that make it useful for identifying prokaryotes are discussed in chapter 10. ■ fluorescence microscope, p. 43, ■ using genotypic characteristics to identify prokaryotes, p. 254

FISH is revolutionizing the study of microbial ecology and holds great promise in clinical laboratories. It provides a means of rapid identification of microorganisms directly in a specimen, bypassing the need to grow organisms in culture. FISH can be used to observe cells of either a specific species or a group of related organisms, depending on the nucleotide sequence of the probe employed. For example, FISH can be used to establish the relative proportions of Bacteria and Archaea in a soil sample by using two separate probes, each specific for one domain, that have different colored fluorescent labels. Another use is to identify and enumerate Mycobacterium tuberculosis cells in a sputum specimen. ■ fluorescence microscope, p. 43, ■ studying microbial ecology, p. 769

Match dark spots with original plate

Figure 9.7 The Steps of a Colony Blot This technique is used to determine which colonies on a plate contain a given DNA sequence. In this example, the probe is labeled with a radioactive isotope, which can be detected using autoradiography.

A less apparent but equally important use of the Southern blot is to distinguish different strains of a given species by detecting subtle variations in their nucleotide sequences. Certain mutations will create, others will destroy, restriction enzymes recognition sequences at particular sites in the genome. Thus, when genomic DNA of different strains is digested with the same restriction enzyme, each will give rise to a slightly different assortment of restriction fragment sizes (figure 9.9). The difference is called a restriction fragment length polymorphism (RFLP) and is the basis for some methods of DNA fingerprinting (see Perspective 9.1). Southern blot hybridization is used to selectively visualize restriction fragments that often vary in size. The probe that is employed is one that has already been shown by trial and error to hybridize to fragments that demonstrate maximal differences among various strains.

Fluorescence in situ Hybridization (FISH)

Fluorescence in situ hybridization (FISH) uses a fluorescently-labeled probe to detect specific nucleotide sequences within intact cells affixed to a microscope slide. After steps that allow probe molecules to hybridize, unbound probe is washed off. Cells within which the probe hybridized can then be viewed

Nucleotide Array Technologies

Nucleotide array technologies employ a microarray, a solid support that contains a fixed pattern of numerous different single-stranded nucleic acid fragments of known sequences (figure 9.10). One of the most sophisticated types of arrays is a DNA chip, a silicon chip of less than an inch in diameter that can carry an array of tens or hundreds of thousands of oligonu-cleotides, short DNA fragments. The power of these arrays is that each nucleic acid fragment functions in a manner analogous to a probe, enabling a researcher to screen a single sample for a vast range of different sequences simultaneously. Unlike typical probes, however, the arrays do not carry a detectable label. Instead, the label must be attached to the nucleic acid of interest—for example, genomic DNA from a bacterium. The DNA of interest is digested into small fragments, denatured, and then added to the array, where it will hybridize to complementary sequences. After washing the array to remove unhybridized DNA, the locations of the labeled DNA molecules are detected. Because the sequence of each of the fragments that make up the array is known, the location of the label can be used to determine the presence of specific sequences in the DNA of interest.

Nucleotide array technology can also be used to study gene expression in those organisms whose genome has been sequenced. Using the information gleaned from the DNA sequence, a microarray can be constructed to contain an oligonucleotide specific for each gene of that particular organism. mRNA isolated from a culture grown under a particular set of conditions and then labeled with a fluorescent marker is allowed to hybridize to the array. The location of the hybridized mRNA reveals the genes that are transcribed under

Organism A 1

Organism B 1

Organism C i

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