• M. Palkina Institute of Animal Breeding and Genetics nd. a. M.V. Zubets of NAAS (Chubynske, Ukraine)
  • O. Metlitska Institute of Animal Breeding and Genetics nd. a. M.V. Zubets of NAAS (Chubynske, Ukraine)
Keywords: DNA, « DNA Sorb - B», «Chelex® -100», proteinase K, exuviae, extinction


The aim of the research – adaptation, optimization and using of existing DNA extraction methods from bees’ biological material with the reagent «Chelex-100" under complex economic conditions of native laboratories, which will optimize labour costs and improve the economic performance of DNA extraction protocol.

Materials and methods. In order to conduct the research the samples of honey bees’ biological material: queen pupae exuviae, larvae of drone brood, some adult bees’ bodies (head and thorax) were selected. Bowl and drone brood were obtained from the experimental bee hives of Institute of Apiculture nd. a. P. I. Prokopovich of NAAS. DNA extraction from biosamples of Apis mellifera ssp. was carried out using «Chelex-100®» ion exchange resin in different concentrations and combinations. Before setting tests for determination of quantitative and quality indexes, dilution of DNA samples of the probed object was conducted in ratio 1:40. The degree of contamination with protein and polysaccharide fractions (OD 260/230), quantitative content of DNA (OD 260/280) in the extracted tests were conducted using spectrophotometer of «Biospec – nano» at the terms of sample volume in 2 µl and length of optical way in 0,7 mm [7]. Verification of DNA samples from biological material of bees, isolated by «Chelex-100®», was conducted after cold keeping during 24 hours at 20°C using PСR with primaries to the fragment of gene of quantitative trait locus (QTL) Sting-2 of next structure [8]:

 3' – CTC GAC GAG ACG ACC AAC TTG – 5’; 3' – AAC CAG AGT ATC GCG AGT GTT AC – 5’ Program of amplification: 94 °C – 5 minutes – 1 cycle; 94 °C – 1 minute, 57°C – 1 minute, 72 °C – 2 minutes – 30 cycles; elongation after 72°C during 2 minutes – 1 cycle. The division of obtained amplicons was conducted by gel electrophoresis at a low current – 7 µÀ, in 1,5 % agarose gel (Sigma ®) in TAE buffer [7].

The results. At the time of optimization of DNA isolation methods, according to existing methods of foreign experts, it was found optimal volume of ion exchange resin solution was in the proposed concentration: instead of 60 µl of solution used 120 µl of «Chelex-100®», time of incubation was also amended from 30 minutes to 180 minutes [9]. The use of the author's combination of method «Chelex-100®» with lysis enzymes, proteinase K and detergents (1M dithiothreitol), as time of incubation was also amended, which was reduced to 180 minutes instead of the proposed 12 hours [10]. Changes in quality characteristics of obtained DNA in samples after reduction in incubation time were not found.

Conclusions. The most economical method of DNA isolation from bees’ biological material is 20% solution of «Chelex-100» ion exchange resin with the duration of the incubation period of 180 minutes. It should also be noted that the best results can be obtained from exuviae, selected immediately after the queen’s exit from bowl, that reduces the likelihood of DNA molecules destruction under the influence of nucleases activation, but not later than 12 hours from release using the technology of isolated obtain of queens.


1. Mathew, C. G. The isolation of high molecular weight eukaryotic DNA. Methods Mol Biol. 2(1985):31–34.
2. Doyle, J. J., and J. L. Doyle. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin. 19(1987):11–15.
3. Kornienko, I. V., D. I. Vodolazhskiy, V. P. Veyko, V. V. Scherbakov, and P. L. Ivanov. 2001. Podgotovka biologicheskogo materiala dlya molekulyarno-geneticheskih identifikatsionnyih issledovaniy pri massovom postuplenii neopoznannyih tel. – Preparation of biological material for molecular-genetic identification surveys at mass admission of unidentified bodies. Rostov-na-Donu, OOO «Rostizdat», 256 (in Russian)
4. Sokolov, B. P., and V. V. Dzhemelinskiy. 1989. Vyidelenie vyisokomolekulyarnoy eukarioticheskoy DNK s ispol- zovaniem atsetata kaliya – Isolation of high molecular weight eukaryotic DNA using potassium acetate. Molekulyarnaya genetika, mikrobiologiya i virusologiya – Molecular Genetics, Microbiology and Virology. (6):45–46.
5. Walsh, P. S., D. A. Metzger, and R. Higuchi. Chelex 100 as a Medium for Extraction of DNA for PCR – Based Typing from Forensic Material BioTechniques. 10(1991):506-509.
6. Evans, J. D., Ryan S. Schwartz, Yan Ping Chen, Gilles Budge, Robert S. Cornman, Pilar De la Rua, Joachim R. de Miranda, Sylvain Foret, Leonard Foster, Laurent Gauthier, Elke Genersch, Sebastian Gisder, Antje Jarosch, Robert Kucharski, Dawn Lopez, Cheng Man Lun, Robin F A Moritz, Ryszard Maleszka, Iren Munoz and M Alice Pinto. 2013. Standart methods for molecular research in Apis mellifera. Jornal of Apicultural Research. 52(4):152–154.
7. Maniatis, T., E. Frich, and Dzh. Sembruk. 1984. Metody geneticheskoy inzhenerii. Molekulyarnoe klonirovanie. Moskow, Mir, 478.
8. Ernesto Guzman-Novoa, Greg J. Hunt, Jose L. Uribe, Christine Smith, and Miguel E. Arechavaleta. 2002. Velasco Confirmation of QTL Effects and Evidence of Genetic Dominance of Honeey Bee Defensive Behavior: Results of Colony and Individual Behavioral Assays. Behavior Genetics. 32(2):95–102.
9. Gregory, P. G., and T. E. Rinderer. 2004. Non – destructive sources of DNA used to genotype honey bee (Apis mellifera) queens. The Netherlands Entomological Society. Entomologia Experimentalis et Applicata. 3:173–177.
10. Songkun Su, Stefan Albert, Shaowu Zhang, Sven Maier, Shenglu Chen, Honghu Du, and Jurgen Tautz. 2007. Non – destructive genotyping and genetic variation of fanning in a honeey bee colony. Journal of Insect Phisiology. 53:411–417.
How to Cite
Palkina, M., & Metlitska, O. (2016). OPTIMIZATION OF DNA EXTRACTION METHODS FROM BIOLOGICAL MATERIALS OF HONEY BEES IN A DIFFERENT METAMOTPHOSIS PHASE. Animal Breeding and Genetics, 52, 171-176. https://doi.org/10.31073/abg.52.22