Quantitative discrimination of 16 S rRNA genes of Dehalococcoides species by MagSNiPer, a quantitative single-nucleotide-polymorphism genotyping method.
Previously we developed MagSNiPer, an SNP (single nucleotide polymorphism) genotyping method. In the present paper we show development of an automated system for MagSNiPer, namely MagSNiPer Station, and its application for quantitative discrimination of Dehalococcoides species, which perform anaerobic dechlorination of chloroethenes. MagSNiPer Station is equipped with a thermal cycler, a tip stand, a microtitre-plate automated stacker, an eight-channel tip dispenser, a magnetic separation unit for Magtration technology, and a chemiluminescence detector. It can automatically perform all processes required for SNP genotyping by MagSNiPer. A primer was designed for discriminating single nucleotide difference between 16 S rRNA genes of Dehalococcoides ethenogenes and Dehalococcoides BAV1. Chemiluminescence intensities for the 16 S rRNA genes obtained by MagSNiPer were proportional to their quantity. MagSNiPer analysis of 16 S rRNA genes amplified on the DNA purified from groundwater gave a ratio of these two 16 S rRNA genes similar to that obtained by cloning and sequencing. MagSNiPer is much easier, more rapid and more cost-effective than conventional sequencing. Compared with denaturing gradient-gel electrophoresis, MagSNiPer has the advantage of being quantitative. Therefore, by applying MagSNiPer at several sites where single base differences exist among Dehalococcoides species, it is possible to analyse Dehalococcoides consortia with ease, yielding useful information on anaerobic bioremediation of chloroethenes.
Yohda
Masafumi
M
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan. yohda@cc.tuat.ac.jp
Kobayashi
Ai
A
Ohishi
Shoji
S
Ebisawa
Maiko
M
Sakihara
Sakari
S
Kakihara
Fumiko
F
Tojo
Yuriko
Y
Tajima
Hideji
H
Nishimura
Minoru
M
D041862:Chloroflexi / Q000235:genetics*; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D013045:Species Specificity
D012336:RNA, Ribosomal, 16S