The study of genetic information carriers—DNA and RNA—depends to a large degree on the possibility of identifying sequences of these materials; that is, determining which sequences are similar or identical, and which are dissimilar.
A complete chemical analysis of each molecule would be the most satisfactory method, but such an analysis, although possible, is very laborious, and it needs a large amount of identical material and thus is not suitable as a first approach to the goal.
Fortunately another much simpler, and very efficient, method has been discovered—the method of molecular hybridization. In the first instance this method leads not to the recognition of similar (or identical) sequences, but to the recognition of complementary sequences. In the double-stranded molecule of DNA the two strands are held together because the bases in each strand have a corresponding, complementary base in the other strand – adenine being complementary to thymidine, and guanine being complementary to cytosine.
If the two strands were to be separated and then brought together, they would reunite by each base finding a complementary base in the other strand. If, instead of bringing together the two strands originally separated from each other, one of the strands is associated with a strand of different origin but containing the complementary bases in the same order as in the original strand, such strands could unite. Furthermore, any strand that can unite with a given strand would be proved to be identical to any other strand that has the same capacity for joining the original or “test” strand.
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The separation of two strands of DNA can be achieved by heating a solution containing DNA to above 65-70° C. On cooling the solution below this temperature, strands of DNA start joining together if they contain complementary sequences of nucleotides, in a process called renaturation.
Molecules of RNA may have sequences of nucleotides which are complementary to the sequences in the DNA as, for instance, in the case of RNA synthesized on a DNA template (except that a uridine nucleotide in the RNA is complementary to the adenine nucleotide in the DNA). Thus, DNA-RNA duplexes may be produced by this method.
If molecules of DNA and RNA join together, the term molecular hybridization is used for this process. Again, there is here a possibility of recognizing similar sequences both in DNA and RNA; all DNA sequences which hybridize with the same RNA sequence must be recognized as identical or at least similar, and all RNA sequences which hybridize with the same DNA sequence must be identical or similar among themselves.
Once the complementary strands have become associated, these can be separated from the single strands (the ones that for one reason or another did not find a suitable partner) by one of two methods. The first method is to use an enzyme, a nuclease, which specifically degrades single-stranded DNA (the S1 nuclease, obtained from Aspergillus oryzae).
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All single-stranded DNA molecules become destroyed, and only the double- stranded fragments remain intact. In the second method the property of hydroxyapatite (calcium phosphate) of binding double-stranded but not single-stranded DNA is exploited.
The DNA fragments in 0.12 M sodium phosphate buffer at pH 6.8 are passed through a column containing hydroxyapatite which binds the duplexes containing more than 50 base pairs, whereas single-stranded material is not absorbed, and passes through. The duplexes are then eluted (washed out) with 4 M phosphate buffer.
The amount of re-association of DNA or DNA/RNA molecules depends mainly on two factors. Firstly, it depends on the concentration of the two complementary sequences—the more molecules that are in solution, the greater the chances for two complementary molecules to meet, an obvious prerequisite for their joining together.
Secondly, the reaction depends on the time during which the reaction is allowed to proceed—the longer the time, the greater the number of complementary molecules that have a chance to collide and join together. These two factors are reflected in a parameter which has been introduced to characterize the dynamics of the re-association of nucleic acid molecules – the Cot, which is simply the product of concentration of the nucleotides in moles per liter by time in seconds.
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The process of re-association can be represented by plotting the Cot on the abscissa, and the proportion of the nucleotides that have formed duplexes on the ordinate. As the value of Cot varies over several orders of magnitude, these values are most conveniently plotted on a logarithmic scale.
The result of such plotting is, in the case of a homogeneous population of nucleotide sequences (that is, when all the sequences in the genome are present in only a single copy), a simple sigmoid curve. In interpreting the curve it must be borne in mind that the curve does not represent the progress of the reaction in time, as the same value for the amount of re-association may be reached, owing to either a greater initial concentration or the reaction having been run for a longer time.
In the case of heterogeneous populations of polynucleotides, which are present in different concentrations the curve is not simple; different groups of molecules associate at different Cot values, and correspondingly the curve may have several rising sections.
The knowledge of the dynamics of molecular hybridization allows the use of this method for determining some quantitative characteristics of certain DNA and RNA sequences. A rare sequence that is present only in low concentrations in any sample will need a much longer time to form duplexes because the chances of such molecules colliding with a complementary partner are small; a sequence that is in great abundance in a sample will re-associate rapidly, because the chances are greater that such molecules will collide with a suitable partner.
Thus the hybridization experiment can give an estimate of the numbers of certain types of molecule in a sample. Furthermore, the method can be used for separation in a mixture of the sequences that are abundant, or common, from the sequences that are rare. It is only necessary to run the reaction for a sufficient time for the common sequences to associate, and then separate them from the molecules remaining single on a hydroxyapatite column.
It was discovered through the use of the molecular hybridization method that in the genomes of eukaryotes some sequences of the DNA are present in multiple copies. As it is not possible at this time to separate particular sections of the DNA performing different functions (sections representing specific genes, or other functional units), recourse has been made to breaking up the DNA from the chromosomes randomly into fragments.
This is done either by using ultrasound, or by squirting a solution of DNA under high pressure through a narrow orifice, or by high-speed blending. The chromosomal DNA is thus broken up into fragments about 500 nucleotides in length. These are then dissociated into a single-stranded condition, and allowed to re-associate.
It is found that some fragments associate very rapidly, showing that they all contain similar (complementary) sequences of nucleotides, while other fragments take a very long time to re-associate, showing that the nucleotide sequences in these fragments were not repeated in the same genome.