A rapid method for identifying diversity within PCR amplicons using a heteroduplex mobility assay and synthetic polynucleotides: application to characterisation of dsRNA elements associated with Cryptosporidium.
A 173-bp fragment of the small extra-chromosomal double-stranded RNA (dsRNA) element of Cryptosporidium parvum was generated by reverse transcriptase PCR from nucleic acid extracted from whole faeces of 18 epidemiologically unrelated cases of cryptosporidiosis. Eleven different sequences were detected and two selected as reference DNA in a heteroduplex mobility assay (HMA). Although sequence diversity was detected, this was difficult to characterise because of the similarity in electrophoretic mobility of the homo- and heteroduplex bands. A PCR method was devised to generate synthetic polynucleotides of greater sequence diversity for use in the HMA. The presence of the synthetic 173-bp fragments was enriched by using, as template for the PCR, material excised from the area of the heteroduplex bands in stained electrophoresis gels. Nine novel sequences were generated and evaluated as reference sequences in the HMA. One of these with 20 bp different from the original sequence was selected for use in the HMA for improved resolution of heteroduplex and homoduplex bands and number of patterns easily resolved (nine different patterns corresponding to different DNA sequences). This method may be useful for analysis of DNA where there is limited natural variation or little sequence variation is described.
PHLS Food Safety Microbiology Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK.
Francesca
F
Leoni
Chris I
CI
Gallimore
Jonathan
J
Green
Jim
J
McLauchlin
D000818:Animals; D001483:Base Sequence; D003457:Cryptosporidiosis / Q000382:microbiology; D003458:Cryptosporidium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016785:Cryptosporidium parvum / Q000235:genetics; D014644:Genetic Variation; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012330:RNA, Double-Stranded / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013997:Time Factors
D012330:RNA, Double-Stranded; D016053:RNA, Protozoan