Figure 10.16 A DNA Melting Curve The absorbance (relative absorbance at 260 nm) rapidly increases as double-stranded DNA denatures, or melts.The Tm is the temperature at which 50% of the DNA has melted; it reflects the GC content.
The GC content varies widely among different kinds of bacteria, with numbers ranging from about 22% to 78%. Organisms that are related by other criteria have DNA base compositions that are similar or identical. Thus, if the GC content of two organisms differs by more than a small percent, they cannot be closely related. For example, although Proteus, EEscherichia, and Pseudomonas are all Gram-negative, rod-shaped
10.7 Using Genotypic Characteristics to Classify Prokaryotes 261
organisms, they cannot be closely related because their GC contents are so different. A similarity of base composition, however, does not necessarily mean that the organisms are related, since many arrangements of the bases are possible. For example, although the bacterium Bacillus subtilis and humans both have 40% GC in their DNA, they are obviously vastly different organisms. The genome size and actual nucleotide sequence differ greatly. The wide range of GC content in some groups, such as Bacillus and Pseudomonas, suggests that many organisms in these groups are unrelated to one another and that their members likely will be classified into other genera as more is learned about them.
The extent of nucleotide sequence similarity between two organisms can be determined by measuring how completely single strands of their DNA will hybridize to one another. Just as two complementary strands of DNA from one organism will base-pair, or anneal, so will the similar DNA of a different organism. The extent of hybridization reflects the degree of sequence similarity. DNA from organisms that share many of the same sequences will hybridize more completely than DNA from those that do not. ■ DNA hybridization, pp. 224,235
One method of determining the extent of hybridization utilizes the steps illustrated in figure 10.17. Extensive DNA similarity suggests that two organisms are closely related. Two
The DNA of one organism is subjected to alkaline conditions or heat to denature the double-stranded DNA, separating it into single-stranded fragments.
The resulting single-stranded fragments are affixed onto a filter or membrane.
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