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
‫• يعتمد المبدأ على استخدام سالسل فردية من الشريط‬
‫الوراثي لبناء سلسلة متممة نكليوتيد تلو اآلخر مع مسح‬
‫وتسجيل القاعدة التي استخدمت‬
‫• من عيوبها أنها تقرأ ‪ 500-300‬نكليوتيد أي أقل من‬
‫طريقة سنغر‬
‫• حاليا تستخدم هذه الطريقة باسم ( ‪454 Life‬‬
‫‪ )Sciences‬وتستطيع إنتاج ‪ 400 Mb‬في عشر‬
‫ساعات في الجهاز الواحد‪ ،‬وتستخدم تقنية ‪GS FLX,‬‬
‫‪pyrosequencing 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 fullspectrum (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: 23700 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