The DNA double helix consists of two strands of nucleotides twisted around each other and held together by hydrogen bonding between the bases. If a solution of DNA is heated, the input of energy makes the molecules vibrate and the hydrogen bonds start coming apart.
If the temperature is high enough, the DNA comes completely apart into two separate strands. This is known as denaturation or “melting”. Since the GC base pair has three hydrogen bonds compared to 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 halfway point on 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. 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 strand will recognize partners by base pairing and double stranded DNA will re-form. This is referred to as annealing. For proper annealing, DNA must be cooled slowly to allow time for single strand to find the correct partners.
Consider two completely different. DNA molecules. If they are mixed, melted and then cooled to re-anneal the single strand, each single strand will recognize and pair with its original complementary strand (Fig. 3.10).
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 formation of hybrid DNA molecules.
The followings are uses of Nucleic acid hybridization:
(a) To Test the Frequency of Relatedness between Two DNA Molecules:
To do this, a sample of first DNA molecule is heated to melt it into single-stranded DNA. The single strands are then attached to suitable filter. Next, the filter is treated chemically to block any remaining sites that would bind DNA. Then, after melting, a solution of second DNA molecule is poured through the filter (Fig. 3.11).
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. The more closely related the two molecules are, the more hybrid molecules will be formed and the higher the proportion of molecule No. 2 will bound by the filter.
For example, if the DNA for a human gene, such as haemoglobin, is 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.
(b) To Isolate Genes for the Process of Cloning:
Suppose we already have the human haemoglobin 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 into short segments with a suitable restriction enzyme.
The gorilla DNA is heated to be melted into single strands and poured over the filter. The DNA fragment that carries the gorilla gene for haemoglobin will bind to human haemoglobin gene and remain stuck to the filter. Other unrelated genes will not hybridize. This approach allows isolation of new genes provided a related gene is available for hybridization.
(c) To Search for Identical or Similar Sequences in Experimental Sample of Target Molecules:
A wide range of method 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 labelled by radioactivity or fluorescence for ease of detection.
The probe is used to search for identical or similar sequences in experimental sample of target molecules. Both the probe and target DNA must be treated to give single-stranded DNA molecules that can hybridize to each other by base pairing. In previous example, the probe DNA would be the human haemoglobin DNA since the sequence is already known. The gorilla DNA would be sample of target molecules.