Sequenase 2

DNA-probe from the pericentromeric region (PCPAflaCEN) of the largest autosome of A. flavicollis specimen #1851 was generated by metaphase chromosome microdissection followed by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) with Sequenase 2.0 (ThermoFisher Scientific, Waltham, MA, USA) according to standard protocol [39 (link),45 ,46 (link)]. Briefly, the chromosome stained with Giemsa (Sigma-Aldrich, Saint Louis, MO, USA) was dissected with an extended glass needle controlled with micromanipulator MR (ZEISS, Jena, Germany) ) on an inverted microscope AXIOWERT10; one copy of the pericentromeric region of the largest autosome was transferred into the extended and siliconized tip of Pasteur pipet with 40 nL collection solution, containing 30% glycerol, 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.1% SDS, 0.1% Triton X-100, 500 μg/mL proteinase K (Boehringer Mannheim, Indianapolis, IN, USA). Dissected material was treated for two hours in a moist chamber at 60 °C and then transferred into 5 µL of PCR mixture for DOP-PCR, which was performed as described [46 (link),47 (link)]. DNA labeling was carried out with 20 additional cycles of PCR performed with an additional fluorescent labeled nucleotide Alexa Fluor 488-5-dUTP (Invitrogen, Waltham, MA, USA) [46 (link),48 (link)].