HotSHOT Plus ThermalSHOCK, a new and efficient technique for preparation of PCR-quality mite genomic DNA.
The present study adapted the HotSHOT method, a technique which has been successfully applied on different kinds of tissues, to studies of Sarcoptes. Some modifications of this technique were made which allowed the quick preparation of PCR-quality Sarcoptes genomic DNA (gDNA), namely applying sodium hydroxide as a substrate for three cycles of thermal shock, followed by a short incubation and pH adjustment with a Tris solution (HotSHOT Plus ThermalSHOCK). The performance of this technique was tested by amplifying a approximately 450-bp rDNA fragment of the second internal transcribed spacer (ITS-2) and by multi-locus genotyping using ten microsatellites on 520 individual Sarcoptes samples. No difference in performance was observed between gDNA samples prepared using the HotSHOT Plus ThermalSHOCK technique and those prepared using a commercial kit utilizing proteinase K digestion. The results demonstrated that the HotSHOT Plus ThermalSHOCK technique is time-saving, economic, and easily automatable for the preparation of PCR-quality mite gDNA, which has implications for studying the molecular biology of mites with human and animal health significance. Although tested in the present study using Sarcoptes mites as a model, this technique may find broad applicability in extraction of gDNA from other parasites with small sizes and hard bodies.
Alasaad
S
S
Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus Las Lagunillas, s. n., 23071 Jaén, Spain.
Rossi
L
L
Maione
S
S
Sartore
S
S
Soriguer
R C
RC
Pérez
J M
JM
Rasero
R
R
Zhu
X Q
XQ
Soglia
D
D
D000818:Animals; D000835:Animals, Wild / Q000469:parasitology; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000235:genetics; D005060:Europe; D016678:Genome; D006358:Hot Temperature; D008924:Mite Infestations / Q000469:parasitology / Q000662:veterinary; D008925:Mites / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic
D017931:DNA Primers; D004275:DNA, Ribosomal; D011933:Reagent Kits, Diagnostic; D004247:DNA