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
| Autor: | Jim McLauchlin, Chris I Gallimore, Jonathan Green, Francesca Leoni |
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| Rok vydání: | 2003 |
| Předmět: |
Microbiology (medical)
Time Factors Molecular Sequence Data Cryptosporidiosis Cryptosporidium Heteroduplex Analysis Microbiology Polymerase Chain Reaction DNA sequencing law.invention chemistry.chemical_compound law Animals Humans Molecular Biology Polymerase chain reaction RNA Double-Stranded Cryptosporidium parvum biology Base Sequence Genetic Variation Reproducibility of Results Sequence Analysis DNA Amplicon biology.organism_classification Molecular biology Reverse transcription polymerase chain reaction chemistry Mutation Nucleic acid DNA RNA Protozoan Heteroduplex |
| Zdroj: | Journal of microbiological methods. 54(1) |
| ISSN: | 0167-7012 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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