Under normal conditions, the two strands of a DNA double helix are held together by hydrogen bonds between the complementary base pairs (see Figure 4–3). But these relatively weak, noncovalent bonds can be fairly easily broken. Such DNA denaturation will release the two strands from each other, but does not break the covalent bonds that link together the nucleotides within each strand. Perhaps the simplest way to achieve this separation involves heating the DNA to around 90°C. When the conditions are reversed—by slowly lowering the temperature— the complementary strands will readily come back together to re-form a double helix. This hybridization, or DNA renaturation, is driven by the re-formation of the hydrogen bonds between complementary base pairs (Figure 8–33). We saw in Chapter 5 that DNA hybridization underlies the crucial process of homologous recombination (see Figure 5–47).
This fundamental capacity of a single-stranded nucleic acid molecule, either DNA or RNA, to form a double helix with a single-stranded molecule of a complementary sequence provides a powerful and sensitive technique for detecting specific nucleotide sequences. Today, one simply designs a short, single-stranded DNA molecule (often called a DNA probe) that is complementary to the nucleotide sequence of interest. Because the nucleotide sequences of so many genomes are known—and are stored in publicly accessible databases—designing a probe to hybridize anywhere in a genome is straightforward. Probes are single-stranded, typically 30 nucleotides in length, and are usually synthesized chemically by a commercial service for pennies per nucleotide. A DNA sequence of 30 nucleotides will occur by chance only once every 1 × 1018 nucleotides (430); so, even in the human genome of 3 × 109 nucleotide pairs, a DNA probe designed to match a particular 30-nucleotide sequence will be highly unlikely to hybridize—by chance— anywhere else on the genome. This, of course, presumes that the sequence complementary to the probe does not occur multiple times in the genome, a condition that can be checked beforehand by scanning the genomic sequence in silico (using a computer) and designing probes that match only one spot. The hybridization conditions can be set so that even a single mismatch will prevent hybridization to “near-miss” sequences. The exquisite specificity of nucleic acid hybridization can be easily appreciated by the in situ (Latin for “in place”) hybridization experiment shown in Figure 8–34. As we will see throughout this chapter, nucleic acid
Figure 8–34 In situ hybridization can be used to locate genes on isolated chromosomes. Here, six different DNA probes have been used to mark the locations of their complementary nucleotide sequences on human Chromosome 5, isolated from a mitotic cell in metaphase (see Figure 4–59 and Panel 17–1, pp. 980–981). The DNA probes have been labeled with different chemical groups (see Figure 8–26B) and are detected using fluorescent antibodies specific for those groups. The chromosomal DNA has been partially denatured to allow the probes to base-pair with their complementary sequences. Both the maternal and paternal copies of Chromosome 5 are shown, aligned side by side. Each probe produces two dots on each chromosome because chromosomes undergoing mitosis have already replicated their DNA; therefore, each chromosome contains two identical DNA helices. The technique employed here is nicknamed FISH, for fluorescence in situ hybridization. (Courtesy of David C. Ward.)
Figure 8–33 A molecule of DNA can undergo denaturation and renaturation (hybridization). For two single-stranded molecules to hybridize, they must have complementary nucleotide sequences that allow base-pairing. In this example, the red and orange strands are complementary to each other, and the blue and green strands are complementary to each other. Although denaturation by heating is shown, DNA can also be renatured after being denatured by alkali treatment.