Single T = stranded melting DNA
Single T = stranded melting DNA
Heating of the DNA double helix melts it into single strands.
into two separate strands (Fig. 21.29A). This is known as denaturation or "melting". Since the GC base pair has three hydrogen bonds compared to the two holding AT together, GC base pairs are stronger than AT base pairs. Therefore, as the temperature rises, AT pairs come apart first and regions of DNA with lots of GC base pairs melt at higher temperatures.
The melting temperature of a DNA molecule is defined as the temperature at the halfway point on the melting curve. The halfway point is used because it is more accurate than trying to guess precisely where melting is complete. Melting is followed by measuring the UV absorption, since disordered DNA absorbs more UV light denaturation When describing proteins or other biological polymers, refers to the loss of correct 3-D structure melting When used to describe DNA, refers to its separation into two strands as a result of heating melting temperature The temperature at which the two strands of a DNA molecule are half-way unpaired
Single strands will base pair to recreate a double helix if slowly cooled together.
Hybrid double helices may be formed by annealing single strands that are related in sequence.
Probes are labeled molecules of DNA (or RNA) that are used to detect complementary sequences by hybridization.
(Fig. 21.29B). Overall, the higher the proportion of GC base pairs, the higher the melting temperature of a DNA molecule.
If denatured DNA is cooled again, the single DNA strands will recognize their partners by base pairing and double stranded DNA will re-form. This is referred to as annealing. For proper annealing, the DNA must be cooled slowly to allow the single strands time to find the correct partners. Consider two completely different DNA molecules. If they are mixed, melted and then cooled to re-anneal the single strands, each single strand will recognize and pair with its original complementary strand (Fig. 21.30). Suppose on the other hand, two closely related DNA molecules are used. Although the sequences may not match perfectly, nonetheless, if they are similar enough, some base pairing will occur. The result will be the formation of hybrid DNA molecules.
The formation of hybrid DNA molecules has a wide variety of uses. For example, how closely two DNA molecules are related may be tested. To do this, a sample of the first DNA molecule is heated to melt it into single-stranded DNA. The single strands are then attached to a suitable filter. Next, the filter is treated chemically to block any remaining sites that would bind DNA. Then, after melting, a solution of the second DNA molecule is poured through the filter (Fig. 21.31). Some of the single strands of DNA molecule No. 2 will base pair with the single strands of DNA molecule No. 1 and will stick to the filter. (As discussed above, DNA molecule No. 2 must be labeled by radioactivity, fluorescence or some other way to enable its detection.) The more closely related the two molecules are, the more hybrid molecules will be formed and the higher the proportion of molecule No. 2 that will be bound by the filter. For example, if the DNA for a human gene, such as hemoglobin, was fully melted and bound to a filter, then DNA for the same gene but from different animals could be tested. We might expect gorilla DNA to bind strongly, frog DNA to bind weakly and mouse DNA to be intermediate. [Several variants of nucleic acid hybridization are in use. This version, involving the binding of DNA to DNA is known as Southern blotting—see below.]
Another use for hybridization is in isolating genes for cloning. Suppose we already have the human hemoglobin gene and want to isolate the corresponding gorilla gene. First the human DNA is bound to the filter as before. Then gorilla DNA is cut it into short segments with a suitable restriction enzyme (see Ch. 22 for details). The gorilla DNA is heated to melt it into single strands and poured over the filter. The DNA fragment that carries the gorilla gene for hemoglobin will bind to the human hemoglobin gene and remain stuck to the filter. Other, unrelated genes will not hybridize. This approach allows the isolation of new genes provided a related gene is available for hybridization.
A wide range of methods based on hybridization is used for analysis in molecular biology. The basic idea in each case is that a known DNA sequence acts as a "probe." Generally the probe molecule is labeled by radioactivity or fluorescence for ease of detection. The probe is used to search for identical or similar sequences in the experimental sample of target molecules. Both the probe and the target DNA must be treated to give single-stranded DNA molecules that can hybridize to each other by base pairing. In the previous example, the probe DNA would be the human hemoglobin DNA since the sequence is already known. The gorilla DNA would be the sample of target molecules.
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