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Optimizing Phytoplasma DNA purification for genome analysis.

Tran-Nguyen, Lucy and Gibb, Karen (2007). Optimizing Phytoplasma DNA purification for genome analysis.. Journal of Biomolecular Techniques,18(2):104-112.

Document type: Journal Article
Citation counts: Scopus Citation Count Cited 5 times in Scopus Article | Citations
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IRMA ID 80802555xPUB21
Title Optimizing Phytoplasma DNA purification for genome analysis.
Author Tran-Nguyen, Lucy
Gibb, Karen
Journal Name Journal of Biomolecular Techniques
Publication Date 2007
Volume Number 18
Issue Number 2
ISSN 1524-0215   (check CDU catalogue open catalogue search in new window)
Scopus ID 2-s2.0-34249845135
Start Page 104
End Page 112
Total Pages 9
Place of Publication United States of America
Publisher The assoc of biomolecular resource facilities
Field of Research 0605 - Microbiology
HERDC Category C1 - Journal Article (DEST)
Abstract Genome analysis of uncultivable plant pathogenic phytoplasmas is hindered by the difficulty in obtaining sufficient quantities of phytoplasma enriched DNA. We investigated a combination of conventional enrichment techniques such as cesium chloride (CsCl) buoyant gradient centrifugation, and new methods such as rolling circle amplification (RCA), suppression subtractive hybridization (SSH), and mirror orientation selection (MOS) to obtain DNA with a high phytoplasma:host ratio as the major first step in genome analysis of Candidatus Phytoplasma australiense. The phytoplasma:host ratio was calculated for five different plasmid libraries. Based on sequence data, 90% of clones from CsCl DNA enrichment contained chromosomal phytoplasma DNA, compared to 60% from RCA CsCl DNA and 20% from SSH subtracted libraries. Based on an analysis of representative libraries, none contained plant DNA. A high percentage of clones (80-100%) from SSH libraries contained extrachromosomal DNA (eDNA), and we speculate that eDNA in the original DNA preparation was amplified in subsequent SSH manipulations. Despite the availability of new techniques for nucleic acid amplification, we found that conventional CsCl gradient centrifugation was the best enrichment method for obtaining chromosomal phytoplasma DNA with low host DNA content.
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