Capillary-electrophoresis restriction fragement length polymorphism : a new method for poxvirus fingerprinting
Yu Li, Richard Kline, Russell Regnery, Inger Damon
Poxvirus Section, Division of Viral and Rickettsial Diseases, CDC, United States
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Capillary-eletrophoresis fragment length polymorphism (CRFLP) is a new method for pangenomic DNA analysis. CRFLP requires digestion of DNA with two restriction enzymes, selective labeling of the fragment ends with a single fluorescent dideoxyribonucleotide, and separation of the labeled fragments by capillary electrophoresis with reference internal size standards from 60 to 640 bases. To validate the accuracy and reproducibility of this new method, CRFLP analyses of genomic DNA from 8 completely sequenced variola strains, and a 12kb DNA fragment from each of the same strins were compared. The CRFLP results were compared with the results predicted form the variola genomic DNA sequences. All sequence predicted restriction site polymorphisms and the fragment-size polymorphisms were detected. Although observed fragment size differed by 1 to 11 nucleotides from the predicted fragment size, single nucleotide differences in polymorphic fragments can be detected to differentiate strains. The CRFLP method is accurate and highly reproducible, which indicates its utility for DNA fingerprinting and diagnostic applications.