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/Illumina Secuencing• True Single Molecule Sequencing • Ion Torrent

454-Pyrosequencing

فردية • سالسل استخدام على المبدأ يعتمدمتممة سلسلة لبناء الوراثي الشريط من

وتسجيل مسح مع اآلخر تلو نكليوتيداستخدمت التي القاعدة

تقرأ • أنها عيوبها أي 500-300من نكليوتيدسنغر طريقة من أقل

باسم )• الطريقة هذه تستخدم 454حالياLife Sciences ) إنتاج Mb 400وتستطيع

الواحد، الجهاز في ساعات عشر فيتقنية GS FLX, pyrosequencingوتستخدم

platform

Solexa/Illumina Secuencing

The sequencing-by-synthesis process.

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

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

Single Molecule Real Time Sequencing

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

The Illumina genotyping platform

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

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