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Polymerase Chain Reaction
Cycle Sequencing
Nucleic Acid Sequencing Gel
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Two methods have been used. The first method, ribonuclease (RNase) T1 oligonucleotide mapping, used direct cleavage of radiolabelled virus RNA to produce oligonucleotides that could be separated by two-dimensional PAGE. Since RNase T1 only cuts at guanosine residues, only about 50 oligonucleotides of sufficient size could be generated; thus only about 10% of the virus genome could be examined, limiting the usefulness of this assay.
It was the advent, in 1977, of the second method, direct nucleotide sequencing, that revolutionised the study of FMD epidemiology.
In this method, single stranded genomic DNA (or complementary DNA in the case of FMD genomic RNA) is incubated with radiolabelled primers, specific for the region of interest, in the presence of polymerase and deoxynucleotides (dNTPs). Four separate aliquot mixtures are used. Each of four types of di-deoxynucleotide, corresponding to the four types of base, is added additionally each to one of the four dNTP mixtures. Polymerisation occurs and random incorporation of the di-deoxynucleotides, among the normal bases, inhibits further growth of that particular molecule. This procedure thus produces for, each aliquot, a mixture of different length oligonucleotides, each terminated by the same base. The four mixtures are then run as a set of four tracks in a PAGE, and analysis of the relative positions of the bands (corresponding to each oligonucleotide segment) in the four tracks gives directly the sequence of the original section of DNA (nucleic acid sequencing gel).
Within 10 years the technique had become refined enough to be used almost on a routine basis. Subsequent developments, polymerase chain reaction and cycle sequencing, have brought the genetic analysis of FMD viruses into truly routine use.
The complete FMDV genome is approximately 8200 nucleotides in length and therefore smaller regions are usually sequenced and compared. Work with various picornaviruses has shown that only genetic comparison of the capsid-coding regions correlates with antigenically defined serotypes. Since this region of the genome is generally the most variable (whether within antigenic sites or not) it is usually the area of choice.
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