Cloning and Expression of Bst DNA Polymerase I Gene in E. coli BL21
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Abstract:
خلاصه DNA پلیمرازها علاوه بر کاربردشان در همسانه سازی و تصحیح ، در انواع تکنیک های ملکولی مانند تکثیر DNA ، جهش های نقطه ای ، توالی یابی DNA ، انواع مختلف PCR ، LAMP و ...اهمیت دارند. پس از کشف PCR تلاش هایی مبنی بر تشخیص و جداسازی آنزیم های مقاوم به دماهای بالا که توانایی تکثیر موثر DNA در دماهای بالا انجام گرفت. در این پژوهش ، سویه Geobacillus stearothermophilus strain 10 به منظور همسانه سازی ژن رمزگذار Bst DNA Polymerase استفاده گردید. پس از استخراج DNA باکتری ، ژن مورد نظر با استفاده از پرایمر های طراحی شده و با روش PCR تکثیرگردید. همسانه سازی ژن رمزگذار آنزیم در ناقل بیانی pET32a و انتقال آن در باکتری E.coli BL21 صورت گرفت. پس از بیان ژن در میزبان با استفاده از IPTG، پروتئین حاصل با استفاده از ستون های IMAC خالص سازی گردید . برای تعیین کیفی فعالیت، آنزیم نوترکیب در واکنش LAMP به کار گرفته شد. نتایج نشان می دهد آنزیم Bst Polymerase می تواند جایگزین مناسبی برای نوع وارداتی آن باشد. کلمات کلیدی: Bst DNA polymerase ، همسانه سازی، بیان ژن، LAMP, PCR
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Journal title
volume 8 issue 32
pages 41- 46
publication date 2019-12-22
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