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DNA sequencing

DNA sequencing. determining the order of nucleotides in DNA Sanger (di- deoxy ) 600-1000 bp Next-Generation Sequencing 2 nd generation 3 rd generation 454-Pyrosequencing Solexa / Illumina Secuencing True Single Molecule Sequencing Ion Torrent. 454-Pyrosequencing.

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DNA sequencing

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  1. DNA sequencing determining the order of nucleotides in DNA • Sanger (di-deoxy) 600-1000 bp • Next-Generation Sequencing • 2nd generation • 3rd generation • 454-Pyrosequencing • Solexa/IlluminaSecuencing • True Single Molecule Sequencing • Ion Torrent

  2. 454-Pyrosequencing • يعتمد المبدأ على استخدام سلاسل فردية من الشريط الوراثي لبناء سلسلة متممة نكليوتيد تلو الآخر مع مسح وتسجيل القاعدة التي استخدمت • من عيوبها أنها تقرأ 300-500 نكليوتيد أي أقل من طريقة سنغر • حاليا تستخدم هذه الطريقة باسم (454 Life Sciences ) وتستطيع إنتاج 400 Mb في عشر ساعات في الجهاز الواحد، وتستخدم تقنية GS FLX, pyrosequencing platform

  3. Solexa/IlluminaSecuencing The sequencing-by-synthesis process.

  4. True Single Molecule Sequencing • By directly sequencing single molecules of DNA or RNA, Helicos' True Single Molecule Sequencing (tSMS™) and Direct RNA Sequencing (DRS™) technologies significantly increase the speed of sequencing, while also decreasing the cost. • Polymerase and one fluorescently labeled nucleotide (C, G, A or T) are added. • billions of templates

  5. Single Molecule Real Time Sequencing • Single molecule real time sequencing utilizes the zero-mode waveguide (ZMW) • A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template • Each of the four DNA bases is attached to one of four different fluorescent dyes. • SMRT chip contains ~3000 ZMW holes • Each of the ZMW holes produces approximately 1,500 bp (base pair) read lengths at a speed of 10 bp per second

  6. Single Molecule Real Time Sequencing

  7. Ion Torrent • Fastest sequencing workflow • Two hour sequencing run for up to 200bp reads • Fully prep 8 samples in parallel in less than 6 hours

  8. The Illuminagenotyping platform

  9. NanoDrop • The NanoDrop® ND-1000 is a full-spectrum (220-750nm) spectrophotometer that measures 1 ul samples with high accuracy and reproducibility. • Nucleic acid concentration and purity of nucleic acid samples up to 3700 ng/ul (dsDNA) without dilution • Fluorescent dye labeling density of nucleic acid microarray samples • General UV-Vis spectrophotometry

  10. NanoDrop • 1 ul only of sample required * Large dynamic range: 2-3700 ng/ul of dsDNA (manufacturer's specs, this is probably pushing it at both ends) * No dilutions for most samples* No cuvettes or capillaries* 10 second measurement • DNA, RNA, microarray labeling dyes, and proteins

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