Oligonucleotide Mapping

Several hundred bacterial endonucleases, called refuel ion endomu lenses, have been identified and purified from various bacteria Each recognizes a unique short, palindromic sequence of nucleotides (a sequence that reads fhe same backward as forward), usually four to six nucleotide pairs long A given restriction endonuclease cleaves the DMA into a precise number of fragments of precise sizes, determined by the location and fiequency of the particular palindromic sequence it recognizes. These DNA fragments may be separated by gel electrophoresis. Different viruses, even very closely related strains of the same virus, yield characteristically diflerent restriction endonuclease fragment patterns, sometimes called fingerprints or restriction fragment length polymorphisms (RFLPs). These have been invaluable for distinguishing between different species or strains of viruses with large genomes, such as poxviruses or herpesviruses. The order of the fragments can be determined to provide a physical map of the genome. Restriction enzymes can also be used to analyze the molecularly cloned cDNA copies of genes or genomes from RNA viruses, and to locate the specific physical positions of various genetic markers on the viral chromosome.

Before restriction endonuclease mapping and sequencing were widely used, oligonucleotide fingerprinting techniques using T1 ribonuclease provided a fast and powerful procedure for differentiating between different isolates of RNA viruses Because T1 ribonuclease cuts RNA on the 3' side of every G residue, very large numbers of short oligonucleotides are produced; hence, adequate separation of the products requires electrophoresis in one dimension followed by chromatography in the other in order to produce a diagnostic fingerprint.

Was this article helpful?

0 0

Post a comment