DNA extraction and barcode identification of development stages of forensically important flies in the Czech Republic.
Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.
a Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources , Czech University of Life Sciences , Praha , Czech Republic.
Tereza
T
Olekšáková
b Institute of Entomology, Biology Centre of the Academy of Sciences of the Czech Republic , České Budějovice , Czech Republic.
Martina
M
Žurovcová
a Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources , Czech University of Life Sciences , Praha , Czech Republic.
Vanda
V
Klimešová
a Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources , Czech University of Life Sciences , Praha , Czech Republic.
Miroslav
M
Barták
c Police of the Czech Republic , Institute of Criminalistics Prague , Prague , Czech Republic.
Hana
H
Šuláková
D000818:Animals; D018153:Czech Republic; D004247:DNA / Q000302:isolation & purification*; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D004175:Diptera / Q000145:classification* / Q000235:genetics / Q000254:growth & development*; D003576:Electron Transport Complex IV / Q000235:genetics; D053803:Forensic Genetics / Q000379:methods; D019476:Insect Proteins / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA
D019476:Insect Proteins; D011933:Reagent Kits, Diagnostic; D004247:DNA; D003576:Electron Transport Complex IV
Forensic science; cytochrome oxidase subunit I; forensic entomology; molecular markers; species identification