Comparison of Three HLB Pathogen Genomic DNA Enrichment Approaches
The presumed causal agent of citrus Huanglongbing (HLB) , Candidatus Liberibacter cpp. , is not yet cultured; thus no genomic DNA directly purified from the pathogen culture for sequencing. Here we reported on comparison of three genomic subtraction approaches to enrich HLB pathogen genomic DNA through subtraction of the overabundant host genome. The first approach was based on the available biotin-avidin affinity chromatography principle, using biotin-labeled driver (healthy host) DNA to remove them in the tester (infected host) DNA through magnetic Dynabeads Streptavidin. The second and third were newly designed digestion-adaptor procedures, both adding a poly (A) adapter to digested driver DNA to remove them in the tester DNA through magnetic Dynabeads poly (dT)M. The difference was the tester digested by the same enzyme (second approach) or an isoschizomer (third approach). Different cleavage sites of isoschizomers prevented remained healthy genomic DNA in the driver from ligating in the subsequent cloning process. We monitored the DNA quantity during the three processes using spectrophotometry and semi-quantitative PCR with host and Liberibacter primers. The biotin-avidin affinity chromatography could only remove about half of the total host DNA in the infected genome from which the enriched bacteria population appeared not enough for subsequent cloning. The isoschizomer adapter mediated approach, slightly better than another enzyme-adapter method, could remove a majority of host DNA (approximately ninety-six percent) , and resulted in a small amount of enriched DNA that was believed likely to contain a substantial portion of Liberibacter DNA. It was proved by random sequencing of three hundred clones from a library of thirty 384-well plates constructed from the DNA enriched by the isoschizomer-adapter mediated approach.
HLB Pathogen genomic DNA Enrichment approaches
Chunxian Chen Qibin Yu Gmitter Jr FG
University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, FL 33850, USA
国际会议
11th International Citrus Congress(第11届国际柑橘大会)
武汉
英文
1081-1086
2008-10-01（万方平台首次上网日期，不代表论文的发表时间）