مقایسه کیفی و کمی نتایج مربوط به چند روش استخراج DNA از ژنوم مایکوباکتریوم‌های بیماری‌زا و ساپروفیت

نوع مقاله: علمی پژوهشی

نویسندگان

1 دانش‌آموخته کارشناسی ارشد زیست‌شناسی- ژنتیک، دانشکده علوم‌پایه، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.

2 استادیار گروه پاتوبیولوژی، دانشکده دامپزشکی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.

3 استادیار گروه زیست شناسی، دانشکده علوم‌پایه، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.

چکیده

با توجه به­ اینکهنخستینمرحلهجهتانجامآزمایش­­هایمولکولیمانند واکنش زنجیره­ای پلی­مراز (Polymerase Chain Reaction; PCR)داشتن DNA کافیوباکیفیتمی­باشد، هدفازانجاماینمطالعهمقایسهپنج روشمختلفاستخراج  DNAژنومی مایکوباکتریوم­هابه­منظور معرفی بهترین روش بود. بدین منظور از پرگنه­های خالص مایکوباکتریوم اویوم پاراتوبرکلوزیس به­عنوان مایکوباکتریوم بیماری­زا و نیز سویه­ای از یک مایکوباکتریوم ساپروفیت به­نام مایکوباکتریوم فلئی استفاده شد. کمیت و کیفیتDNAهای به­دست­آمده توسط هریکاز روش­ها، به­ترتیب بر اساس غلظت DNAاستخراجی (نانوگرم در میکرولیتر) در طول موج 260 نانومتر و نیز بر مبنای نسبت OD در طول موج­های 280/260 با استفاده از دستگاه­ نانودراپ محاسبه و موردارزیابی قرارگرفت. استخراج DNA از ژنوم مایکوباکتریوم­هابااستفادهازروش CTAB (Cetyltrimethylammonium bromide) بیشترین میزان و روشمبتنی بر جوشانیدن کمترینمیزانDNA را حاصل کرد. همچنین مقایسهمیزانخلوصDNA استخراج­شدهدراینروش­ها نیزنشانداد که استخراج DNA بااستفادهاز روشCTAB بیشترینمیزان وروشمبتنی بر جوشانیدن بازهم کمترین میزانخلوصراداشتهاست. همچنین بین دو نوع باکتری از نظر کیفیت DNAهای استخراج‌شده در هیچ­یک از روش­های استفاده­شده برای استخراج DNA اختلاف معنی­داری وجود نداشت. با توجه به نتایج مطالعه حاضر به­نظر می­رسد که استفادهازروش CTAB یکی از مناسب­ترین روش­ها جهت استخراج DNAژنومیاز مایکوباکتریوم­هاجهتتشخیص مولکولی آنها در نمونه­های مختلفمی­باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Comparing qualitative and quantitative results of some DNA extraction methods from genome of pathogenic and saprophytic mycobacteria

نویسندگان [English]

  • Ali Nasiri 1
  • Younes Anzabi 2
  • Mohammad reza Mashayekhi 3
1 Graduate of Biology-Genetic Master Science, Faculty of Basic Sciences, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
2 Assistant Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
3 Assistant Professor, Department of Biology, Faculty of Basic Sciences, Tabriz Branch, Islamic Azad University, Tabriz, Iran.
چکیده [English]

Given that the first step in performing molecular techniques such as PCR (Polymerase Chain Reaction (is having enough high quality DNA, the aim of this study was to compare five different extraction methods of genomic DNA of mycobacteria in order to introduce the best method. For this purpose, pure colonies of Mycobacterium avium paratuberculosis were used as pathogenic mycobacterium and also a saprophytic strain called Mycobacterium phelei. The quality and quantity of the obtained DNA by each method was calculated, measured and evaluated respectively; based on extracted DNA concentrations (nanograms per microliter) at a wavelength of 260 nm and also optical density (OD) ratio based on the 260/280 wavelength by using the Nanodrop technique. The highest and lowest amounts of mycobacterial genome DNA was extracted using the CTAB (Cetyltrimethylammonium bromide) and boiling methods respectively. Comparison of the purity of extracted DNA also indicated that the highest purity was obtained using the CTAB method and the lowest using the boiling method. Also no significant differences were observed regarding the quality of extracted DNA from pathogenic and saprophytic mycobacteria among the evaluated extraction methods. Thus, according to the results of this study it appears that using the CTAB method is the most suitable way to extract genomic DNA for molecular detection of mycobacteria in different samples.

کلیدواژه‌ها [English]

  • DNA extraction
  • genome
  • Mycobacterium
  • Pathogen
  • Saprophyte
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