Molecular Biology
Dr. Chaim Wachtel
April 4, 2013
Topics
• Microarray
• Deep Sequencing
• PCR
– Real-Time
– Digital
– Nanostring
GENE EXPRESSION
Microarray
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Affymetrix
Agilent
Same idea different implementation
Pros and cons to each method
Agilent
The Chips
Affymetrix
Printing of Affymetrix Chips
Printing of Agilent Chips
Printing of Agilent Chips
Probes are 60 nucleotides in length vs 25 for Affymetrix
Affymetrix workflow
Agilent workflow
Analysis
• Feature Extraction
• Bioinformatics
Agilent array
“home-made”=low-density
Pros and Cons
• Cost
• Dual color vs single
• Specificity
Deep Sequencing
Next Generation Sequencing
(NGS)
Sanger Sequencing
Multiple Platforms
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Illumina
454
Solid
Nanopore/Oxford
Ion Torrent
Different companies different
methods
Workflow for Sequencing
Workflow for Sequencing
• Depends on project- RNA or DNA
• Protocols are ~90% the same for all systems
Illumina
Workflow for Sequencing
• Bridge PCR
• Emulsion PCR
Sequencing with Illumina
Sequencing with Solid
The Ion Torrent
Scalability
The Chip
Nanopore Technology
* all images are of an alpha prototype of
the MinION device, final device
expected to be at least half this size
How does it work?
What’s the big deal??
• GridION, consists of an array of sequencers, each containing
2,000 nanopores.
• That is expected to increase to 8,000 nanopores in the nextgeneration device. Twenty of these, running simultaneously,
could sequence a full human genome in 15 minutes, the company
said, describing both devices as "plug-and-play."
• MinION, with 512 nanopores
• will be capable of sequencing 150,000 DNA bases per hour
• the 48,490-bp genome of lambda DNA was sequenced as a
complete fragment—in one read. This read length is three
orders of magnitude larger than the typical length of DNA
fragments that are read in current sequencing approaches using
fluorescent nucleotide labeling3.
• $$$$- MinION will cost $900