بررسي كارآيي چهار روش استخراج DNA از زنبورعسل كوچك Apis florea (Hymenoptera: Apidae ) جهت انجام آزمايشات ژنتيكي

Evaluation of four different DNA extraction methods from dwarf honeybee Apis florea (Hymenoptera: Apidae) for genetic experiments

استخراج DNA اولين مرحله در انجام آزمايشات ژنتيكي است، لذا كيفيت و كميت مناسب DNA استخراج شده در مطالعات مولكولي امري بسيار ضروري مي باشد. از اين رو دستيابي به روش يا روش ­هايي كه به سهولت استخراج DNA از زنبورعسل كوچك Apis florea Fabricius كه يكي از دو گونه زنبورعسل موجود در ايران است كمك كند، مي ­تواند حائز اهميت باشد. اين گونه زنبورعسل به دليل پراكندگي گسترده و همچنين ايفاي نقش مستقيم در گرده افشاني گونه‌هاي زراعي، باغي و جنگلي از اهميت مطالعاتي زيادي برخوردار است. به همين دليل در اين مطالعه چهار روش استخراج DNA شامل بهينه نمكي، فنل-كلروفرم، CTAB و CTAB+SDS به منظور گزينش مناسب­ ترين روش استخراج DNA زنبورعسل كوچك مورد ارزيابي و آزمايش قرار گرفت. سپس كيفيت و كميت DNA استخراجي با استفاده از روش­ هاي اسپكتوفتومتري و ژل آگارز مورد مقايسه قرار گرفت. با توجه به كميت و كيفيت DNA استخراج شده، روش CTAB+SDS براي استخراج DNA از زنبورعسل كوچك قابل توصيه است.

Background and Objectives DNA extraction is the first step of genetic experiments. In this regard, quality and quantity of extracted DNA plays an important role in molecular studies. This experiment was conducted in order to achieve a novel method that helps us to extract DNA from Apis florea F (one of two important Iranian honeybee). This species is regarded as an important honeybee due to its world-wide distribution and direct role in pollination of field crops, orchards and forests. Materials and Methods Samples were collected randomly from the hives of dwarf honeybee in Khuzestan province. In the current research, DNA was extracted from the thorax tissues of worker bees. Totally, four DNA extraction methods were evaluated including the optimal salting out, phenol-chloroform, CTAB and CTAB+SDS. To evaluate the quality of extracted DNA, spectrophotometry, electrophoresis and agarose gel methods were applied to compare quantity and quality of DNA extraction. Results Quantity and quality of extracted DNA was evaluated by spectrophotometry based on the absorption ratio 260/280 and electrophoresis on agarose gel. Based on the absorption ratio, for the methods of salting out, phenol-chloroform, CTAB and CTAB + SDS were 1.15 ± 0.01, 1.37 ± 0.01, 1.72 ± 0.02 and 1.82 ± 0.01, respectively. Duncan multiple range test revealed that CTAB+SDS was significantly different from other methods for evaluation of DNA extraction. Discussion Based on the results, the quality of DNA visible bands on the agarose gel can be considered as the option to evaluate the quality of DNA. On the other hand, twice washing in the CTAB + SDS method reduced protein contamination significantly. Furthermore, it could be mentioned that all the DNA extraction methods used in this study seems to be suitable in polymerase chain reaction test, but CTAB+SDS resulted in higher DNA quality and quantity. We referred to this thorax tissue as “thoracic muscle mass”, which could be as candidate tissue to obtain larger quantities of DNA.