Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.
This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements. Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.
Proudnikov
D
D
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, IL 60439, USA.
Kirillov
E
E
Chumakov
K
K
Donlon
J
J
Rezapkin
G
G
Mirzabekov
A
A
D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D008856:Microscopy, Fluorescence; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010784:Photomicrography; D017354:Point Mutation; D017955:Poliovirus / Q000235:genetics* / Q000472:pathogenicity; D011055:Poliovirus Vaccine, Oral / Q000009:adverse effects / Q000592:standards; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012367:RNA, Viral / Q000235:genetics*; D015203:Reproducibility of Results; D012449:Safety; D012984:Software; D014774:Virulence / Q000235:genetics
D018076:DNA, Complementary; D011055:Poliovirus Vaccine, Oral; D012367:RNA, Viral