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Freezing Method as a New Non-Destructive Modification of DNA Extraction

Yaakop, S., C. van Achterberg, Idris, A. B. and Aman, A. Z.

Pertanika Journal of Tropical Agricultural Science, Volume 36, Issue 4, November 2013

Keywords: Freezing method, molecular DNA, non-destructive samples, sufficient DNA, phylogenetic analysis.

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A modification of a DNA extraction method by freezing specimens is recognized as one of new non-destructive techniques. In this study, the freezing method has been applied on dried and fresh, tiny and economically important insect samples, i.e. on adults and larvae of wasps, fruit flies and thrips. The modification entails freezing instead of a lengthy incubation of the sample. Most importantly, the sample is not cut into small pieces, but is soaked in a lysis buffer and then frozen in -22°C for a minimum of 20 minutes. After that, the remaining protocols from the manual of DNeasy Blood and Tissue Kit are followed. Several other non-destructive methods also require incubation for at least 20 minutes in a lysis buffer at 55°C. However, the duration of that incubation process is not standard for all insect and arthropod species. This is because the optimization process is based on species size and the thickness of the insect cuticle. With the freezing method, samples are not damaged, and remain available for morphological re-examination. Hence, the sample can also be re-used for taxonomic work with no distortion of samples, no loss of coloration and no phenotypic changes on the external morphology. The complete protocol for the freezing method is described in this paper. With this freezing method, DNA concentration of 0.2- 5.61 ng/µl was recovered on various tiny insect species. Furthermore. several specimens of Bactrocera and Heratemis species were selected as control specimens in analyzing a variety of extraction methods. The freezing method was proven as a new technique to obtain sufficient quantity and a high quality of DNA for molecular work

ISSN 1511-3701

e-ISSN 2231-8542

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