molecular biology dr. chaim wachtel april 4, 2013

Molecular Biology

Dr. Chaim WachtelApril 4, 2013

Topics

• Microarray• Deep Sequencing• PCR

– Real-Time– Digital– NanostringGENE EXPRESSION

Microarray

• Affymetrix• Agilent• Same idea different implementation• Pros and cons to each method

The ChipsAgilent 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

• Feature Extraction• Bioinformatics

Analysis“home-made”=low-density

Agilent array

Pros and Cons

• Cost• Dual color vs single• Specificity

Deep Sequencing

Next Generation Sequencing (NGS)

Sanger Sequencing

Multiple Platforms

• 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

next-generation 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