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Homo sapiens (human)

3,000,000,000

Zea mays (corn)

4,500,000,000

Amphiuma (salamander)

765,000,000,000

times as much DNA as that of human cells. Clearly, these differences in C value cannot be explained simply by differences in organismal complexity. So what is all this extra DNA in eukaryotic cells doing? We do not yet have a complete answer to this question, but examination of DNA sequences has revealed that eukaryotic DNA has complexity that is absent from prokaryotic DNA.

Denaturation and Renaturation of DNA

The first clue that the DNA of eukaryotes contains several types of sequences came from the results of studies in which double-stranded DNA was separated and then allowed to reassociate. When double-stranded DNA in solution is heated, the hydrogen bonds that hold the two strands together are weakened and, with enough heat, the two nucleotide strands separate completely, a process called denaturation or melting ( FIGURE 11.13). DNA is typically denatured within a narrow temperature range. The midpoint of this range, the melting temperature (7m), depends on the base sequence of a particular sample of DNA: G-C base pairs have three hydrogen bonds, whereas A-T base pairs only have two; so the separation of G-C pairs requires more energy than does the separation of A-T pairs. A DNA molecule with a higher percentage of G - C pairs will therefore have a higher Tm than that of DNA with more A-T pairs.

The denaturation of DNA by heating is reversible; if single-stranded DNA is slowly cooled, single strands will collide and hydrogen bonds will again form between complementary base pairs, producing double-stranded DNA (see Figure 11.13). This reaction, called renaturation or reannealing, takes place in two steps. First, single strands in solution collide randomly with their complementary strands. Second, hydrogen bonds form between complementary bases.

Two single-stranded molecules of DNA from different sources will anneal if they are complementary, a process termed hybridization. For hybridization to take place, the two strands do not have to be complementary at all their bases—just at enough bases to hold the two strands together. The extent of hybridization can be used to measure the similarity of nucleic acids from two different sources and is a common tool for assessing evolutionary relationships. The rate at which hybridization takes place also provides information about the sequence complexity of DNA (see next subsection).

Renaturation Reactions and C0t Curves

In a typical renaturation reaction, DNA molecules are first sheared into fragments several hundred base pairs in length. Next, the fragments are heated to about 100°C, which causes the DNA to denature. The solution is then cooled slowly, and the amount of renaturation is measured by observing optical absorbance. Double-stranded DNA absorbs less UV light than does single-stranded DNA; so the amount of renaturation can be monitored by shining a UV light through the solution and measuring the amount of the light absorbed.

The amount of renaturation depends on two critical factors: (1) initial concentration of single-stranded DNA (C0) and (2) amount of time allowed for renaturation (f). Other things being equal, there will be more renaturation rt If a solution of double-stranded DNA is slowly heated, the nucleotide strands separate.

^ If the solution is then cooled, the complementary single strands will come back together (reanneal).

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