Molecular Biology

Dr. Chaim WachtelMay 28, 2015

Topics

• Microarray• Deep Sequencing• PCR

– Real-Time– Digital– NanostringGENE EXPRESSION

Enzymes: Reverse Transcriptase, DNA polymerase, RNA polymerase

Microarray in theory

Microarray in practice

Common Applications

•Gene expression profiling•Chromatin

immunoprecipitation on Chip (ChIP)

•SNP detection•Alternative splicing detection

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)

High Throughput Sequencing (HTS)

Timeline and Comparison of Commercial HTS Instruments

High-Throughput Sequencing Technologies, Molecular Cell, 58, 586–597 ( May, 2015) Jason A. Reuter1, Damek V. Spacek1, Michael P. Snyder

Overview of Selected HTS Applications

High-Throughput Sequencing Technologies, Molecular Cell, 58, 586–597 ( May, 2015) Jason A. Reuter1, Damek V. Spacek1, Michael P. Snyder

In 1958, he was awarded a Nobel Prize in chemistry "for his work on the structure of proteins, especially that of insulin". In 1980,  he shared with Walter Gilbert half of the chemistry prize "for their contributions concerning the determination of base sequences in nucleic acids". The other half was awarded to Paul Berg "for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA"

Who is this? And what is he holding???

Sanger Sequencing

Sanger program

Capillary Sequencing

Machines have 1-96 capillaries

Multiple Platforms

• Illumina• 454• Solid• Nanopore/Oxford• Ion Torrent• Pacific Biosciences

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

Ilumina Machines

Ilumina Machines

Sequencing with Solid

The Ion Torrent

Scalability

The Chip

PacBio and Nanopore

High-Throughput Sequencing Technologies, Molecular Cell, 58, 586–597 ( May, 2015) Jason A. Reuter1, Damek V. Spacek1, Michael P. Snyder

Nanopore Technology

Movie time

• https://vimeo.com/127689053?from=outro-embed

• https://nanoporetech.com/products-services/minion1

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

Pacific Biosystems

• https://www.youtube.com/watch?v=NHCJ8PtYCFc&list=UU2y78sjVOumGc2da1tN629g