Insight into the uniqueness of genetic material among organisms begin in the 1940's, and has continued to the present day by analysis of base ratios, density gradients, melting temperature, nearest neighbor frequencies, and reassociation rates. More direct attempts at basic deciphering the base by base sequence of nucleic acids first bore fruit in 1965 when holy and co worker determined the 77-nucleotide-long sequence of a particular transfer RNA molecule in yeast(tRNA). The technique they used depend on an elegant laborious method that took more than a year to analyze this relatively short nucleotide sequence. However, it was this discovery that led Khorana and coworkers to synthesize a sequence of DNA nucleotides exactly complementary to the tRNA molecule and represented the first laboratory creation of gene that specify a particular cellular product.
Since the time of Holy's work, nucleotide sequence analysis has undergone important changes, and ingenious techniques have been developed by Maxam and Gillbert, and by Sanger and coworkers. These techniques depend on the isolation many uniform repeated lengths of DNA through restriction endonuclease digests and DNA cloning methods. In the Maxam-Gillbert procedure, copies of DNA sequence to be analyzed are labeled at their 5' ends with radioactive 32P, and are than broken at various points by four separate chemical treatments, each treatment removing on the average either one purine or one pyrimidine from any particular chain. The result of each such break in the DNA is to generate a 32P-labeled fragment of a specific length that bands at a specific position on a gel subjected to an electric gradient. This process of gel electrophoresis can separate molecules that differ by only a single nucleotide length and than 32p labelled DNA fragments on the gel can than be detected by auto radiography.
After autoradiography, the four chemical treatments of a particular DNA sequence lead to a series of unique fragments based on the removal of nucleotides containing either G, A,T or C. By absorbing the bands form at a specific position on the electrophoretic gel, one can determine bot the length of fragment and which of the nucleotide removed was responsible for generating the particular fragment. For example, the band at the bottom row indicates a DNA sequence one nucleotide long that was formed as a result the loss of G that is, the gene nucleotide remove must have been at 3' end of the fragment. Similarly, a A was removed to form the dinucleotide fragment on the second row from the bottom etc. Thus the nucleotide sequence of a particular DNA can be reconstructed by identifying the 5' terminal end and than by reading the bands in ascending order. At present, a sequence of 200-400 nucleotides may be determined from a single gel, and the nucleotide sequence of much longer DNA chain can be constructed by sequencing segments that overlap adjacent segments.
Although the sequencing method devised by Sanger is some what different, the result of both these analytical techniques and their modifications has been to produce an enormous amount of essential and interesting data. We now know the entire nucleotide sequences of a number of small viruses approximately 5-6kb long, the human mitochondrial chromosome, as well as the sequences of a variety of fairly long chromosome sections in many other organisms. RNA sequences have also been determined by direct sequencing methods or by converting them into complementary DNA sequences and than analyzing the latter.