Böcek Dokularından DNA İzolasyonu Yöntemlerinin Kalite, Verim ve Maliyet Açısından Karşılaştırılması

Abstract

Günümüzde moleküler biyoloji ve biyoinformatik alanındaki gelişmelere paralel olarak metagenomik, biyoteknoloji, tüm genom dizilemesi, genom evrimi gibi alanlarda çok sayıda araştırma yapılmaktadır. Böcek türleri ile yapılan çalışmalarda ilk ve en önemli adım etkili bir DNA izolasyonu yönteminin kullanılmasıdır. Kitin yapılı bir dış iskelete sahip olan böceklerin çoğunun küçük canlılar olması DNA izolasyonunu zorlaştırmaktadır. Yapılan bu çalışma ile Cephus pygmeus (Linnaeus, 1767) türüne ait bireylerden dokuz farklı DNA izolasyon yöntemi ile total DNA izolasyonu gerçekleştirilmiştir. Elde edilen DNA örnekleri verim, saflık, maliyet, zaman ve PCR başarısı kriterleri altında değerlendirilmiştir. Maliyet ve kullanılan kimyasalların toksisitesi göz önüne alındığında ise tuzla çöktürme yönteminin böcek dokularından DNA izolasyonu için en uygun yöntem olduğu yorumlanmıştır.

 

Keywords

• PCR,DNA İzolasyon Yöntemleri,Böcek Genomik DNA’sı,DNA Kalitesi,Cephus pygmeus

Comparison of DNA Isolation Methods From Insect Tissues in Terms of Quality, Yield and Cost

Abstract

Today, there are many studies conducted in the areas such as metagenomics, biotechnology, whole genome sequencing, genome evolution, and so on parallel to the developments in molecular biology and bioinformatics in molecular biology and bioinformatics. The first and most important step in studies with insect species is the use of an effective DNA isolation method. Small body and external chitin skeleton of the insects cause difficulties in isolating the insect DNAs. In this study, nine different DNA isolation methods were applied to carry out total DNA isolation from the individuals belonging to the species Cephus pygmeus (Linnaeus, 1767). The results were interpreted in terms of DNA yield, purity, cost, time and PCR performance criteria. When the cost and the toxicity of the used chemicals are taken into consideration, it is interpreted that the salting out method is the most suitable method for DNA isolation from insect tissues. 

Keywords

• DNA Isolation Protocols,Insect Genomic DNA,DNA Quality,PCR,Cephus pygmeus

References

1. Aljanabi SM, Martinez I (1997). Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic acids research 25: 4692–4693.
2. Asghar U, Malik MF, Anwar F, Javed A, Raza A (2015). DNA Extraction from Insects by Using Different Techniques: A Review. Advances in Entomology 3: 132–138.
3. Biere A, Bennett AE (2013). Three-way interactions between plants, microbes and insects. Functional Ecology 27: 567–573.
4. Carlton JM, Adams JH, Silva JC, Bidwell SL, Lorenzi H, Caler E, Crabtree J, Angiuoli S V, Merino EF, Amedeo P (2008). Comparative genomics of the neglected human malaria parasite Plasmodium vivax. Nature 455: 757–763.
5. Carpenter JM, Carpenter JM, Wheeler WC, Wheeler WC (1999). Towards simultaneous analysis of morphological and molecular data in Hymenoptera. Zoologica: 251–260.
6. Chen H, Rangasamy M, Tan SY, Wang H, Siegfried BD (2010). Evaluation of five methods for total DNA extraction from western corn rootworm beetles. PLoS ONE 5: e11963.
7. Davey JL, Blaxter MW (2010). RADseq: Next-generation population genetics. Briefings in Functional Genomics 9: 416–423.
8. Van Dijk EL, Auger H, Jaszczyszyn Y, Thermes C (2014). Ten years of next-generation sequencing technology. Trends in Genetics 30: 418–426.

9. Furlong MJ (2015). Knowing your enemies: Integrating molecular and ecological methods to assess the impact of arthropod predators on crop pests. Insect Science 22: 6–19.
10. Gutiérrez-López R, Martínez-de la Puente J, Gangoso L, Soriguer RC, Figuerola J (2015). Comparison of manual and semi-automatic DNA extraction protocols for the barcoding characterization of hematophagous louse flies (Diptera: Hippoboscidae). Journal of Vector Ecology 40: 11–15.
11. Hebert PDN, Ratnasingham S, deWaard JR (2003). Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings. Biological sciences / The Royal Society 270 Suppl: S96–S99.
12. Ji YJ, Zhang DX, He LJ (2003). Evolutionary conservation and versatility of a new set of primers for amplifying the ribosomal internal transcribed spacer regions in insects and other invertebrates. Molecular Ecology Notes 3: 581–585.
13. Juen A, Traugott M (2005). Detecting predation and scavenging by DNA gut-content analysis: A case study using a soil insect predator-prey system. Oecologia 142: 344–352.
14. Korkmaz EM, Budak M, Orgen SH, Bagda E, Gencer L, Ulgenturk S, Basibuyuk HH (2010). New records and a checklist of Cephidae (Hymenoptera: Insecta) of Turkey with a short biogeographical consideration. Turkish Journal of Zoology 34: 203–211.
15. Korkmaz EM, Lunt DH, Çıplak B, Değerli N, Başıbüyük HH (2014). The contribution of Anatolia to European phylogeography: the centre of origin of the meadow grasshopper, Chorthippus parallelus. Journal of biogeography 41: 1793–1805.
16. Kress WJ, García-Robledo C, Uriarte M, Erickson DL (2015). DNA barcodes for ecology, evolution, and conservation. Trends in Ecology and Evolution 30: 25–35.
17. Mardis ER (2017). DNA sequencing technologies: 2006–2016. Nature Protocols 12: 213–218.
18. Misof B, Liu S, Meusemann K, Peters RS, Donath A et al. (2014). Phylogenomics resolves the timing and pattern of insect evolution. Science 346: 763–767.
19. Rosenfeld J, Foox J, DeSalle R (2015). Insect genome content phylogeny and functional annotation of core insect genomes. Molecular Phylogenetics and Evolution 97: 224–232.
20. Shanower TG (2008). History of biological control of wheat stem sawflies (Hymenoptera: Cephidae). In: Capinera J (Ed), Encyclopedia of Entomology, Springer, Netherlands, s. 1826–1829.
21. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994). Evolution, weighting, and phylogenetic utility of mitochondrial gene-sequences and a compilation of conserved polymerase chain-reaction primers. Annals of the Entomological Society of America 87: 651–701.
22. Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML (2000). Preparation of PCR-quality mouse genomic dna with hot sodium hydroxide and tris (HotSHOT). BioTechniques 29: 52–54.
23. Walsh PS, Metzger DA, Higuchi R (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10: 506–513.
24. Wingfield MJ, Klein H (2012). DNA extraction techniques for DNA barcoding of minute gall-inhabiting wasps. Molecular Ecology Resources 12: 109–115.