Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis.
We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.
Konakandla
Bhanu
B
Department of Entomology, Kansas State University, 123 Waters Hall, Manhattan, KS 66506-4004, USA.
Park
Yoonseong
Y
Margolies
David
D
D000818:Animals; D021901:DNA, Intergenic / Q000737:chemistry / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D016678:Genome; D008925:Mites / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D012117:Resins, Synthetic / Q000737:chemistry; D017422:Sequence Analysis, DNA
D021901:DNA, Intergenic; D004272:DNA, Mitochondrial; D012335:RNA, Ribosomal; C075526:RNA, ribosomal, 12S; D012117:Resins, Synthetic; C024997:Chelex 100