A Summary of Some Recent Advances in DNA Sequencing Technology

Sara Caldwell

IRR

2/24/10

Current Sequencing Technique

1. DNA/RNA is isolated from source organism

2. DNA/RNA amplified using PCR/RT-PCR with specific primers

3. PCR products analyzed using gel electrophoresis

4. Purification to remove template, polymerase, etc.

5. Sequenced using dye terminator sequencing

Dye-terminator Sequencing

• DNA polymerase assembles compliment strand

• Reaction terminated by labeled ddNTP

• Fluorescent signal detected by instrument using capillary electrophoresis

Dye-terminator sequencing- cont’d

• Signals measured and recorded in chromatogram• Analysis performed using base-call software

Problems with dye-terminator technique

• Errors or no detection of first 15-40 base pairs• Enzyme dependent• Excess background noise• Tedious analysis with base-call software• Expenses

– PCR kit, primers, purification steps, other analysis

• Time consuming• Single reaction can sequence 300-1000 base pairs

– Inefficient for genomic work

A Need for New Sequencing Strategies

• Faster• Less expensive• More accurate• Capable of handling longer

nucleotides• National Human Genome

Research Initiative

Recent Advances & Publications

• Enzyme based sequencers– Pacific Biosciences SMRT sequencing

• Real Time single molecule fluorescent sequencing• Nanopore based sequencing

– DNA sequences “read” through nanopores– Wanunu et al in Nature Nanotechnology

• Boston University• December 2009• Focused 800-8,000 bp length dsDNA through a

nanopore using a salt gradient– Chang et al in Nano Letters

• Arizona State University• February 2010• Detected individual bases through nanopore capture

Pacific Biosciences SMRT Sequencing

• Single Molecule Real-Time (SMRT)• “Eavesdrops” on a single DNA polymerase

– Replication with phosphate-linked fluorophore labeled dNTPs

– Polymerase assembles long strand of DNA

– 1-3 bases assembled and detected per second

• 20,000 X faster than current sequencing!

How SMRT Works• Uses a nanoscale observation window

– DNA polymerase adhered to transparent base of small metal cylindrical nanotube

– Polymerase assembles compliment strand with phospho-linked bases

– Brief fluorescent flash released when phosphate is cleaved

– Flash detected and generated into sequence data

Nano-scale Observation Windows

Plate with thousands of Plate with thousands of nanoscale cylindersnanoscale cylinders

Individual “windows” Individual “windows”

(~50 nm in diameter)(~50 nm in diameter)

Observation windows for DNA Polymerase

DNA polymerase adhered to transparent glass surface of windowDNA polymerase adhered to transparent glass surface of window

Fluorescence recorded as polymerase assembles complimentary DNA

Fluorescent signal recorded from window with minimal light interferenceFluorescent signal recorded from window with minimal light interference

How the Signal Works- Fluoropores

Traditional base-linked fluoropore Phospho-linked fluoropore(used by SMRT)

Connections Matter…

• High amount of interference with base linked fluorescence– Multiple bases signals sent

simultaneously during assembly

• Low signal noise with phosphate links– Brief flash as polymerase

cleaves base

Base linked interferenceBase linked interference

Flashes detected during complimentary DNA Assembly…

Results…clear, accurate sequencing!

• Can assemble long DNA molecules– >10,000 bp

• Rate of 1-3 bases per second

• Limited by detection speed, not polymerase

• More improvements available– Enhanced polymerases, changing chamber content

to customize assays, etc.

Non-Enzyme based Sequencing Recently Published Advances

• Wanunu, Meni; Will Morrison, Yitzhak Rabin, Alexander Y. Grosberg, Amit Meller.“Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient” Nature Nanotechnology. 20 December 2009.

• Chang, Shuai; Shuo Huang, Jin He, Feng Liang, Peiming Zhang, Shengqing Li, Xiang Chen, Otto Sankey, Stuart Lindsay. “Electronic Signatures of all four DNA nucleosides in a Tunneling Gap” Nano Letters. 2010

Nanopores & Nanotechnology

• New techniques used tiny charged metal nanopores for threading and “reading” DNA with electronic probes

• Requires no enzyme• No PCR amplification

required• Increasing base pair

length capabilities• Cost effective

– Limited number of reagents needed

Threading dsDNA through nanopores…

• “Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient” Nature Nanotechnology. 20 December 2009

• Boston University- lead by Amit Meller

• Threaded 800-8,000 base pair length double stranded DNA (dsDNA) through nanopore

• Also showed that DNA can be detected this way on the picomolar level (no amplification required!)

Focusing Unlabelled DNA into Nanopores

DNA captured in mouth of 5 nm nanoporeDNA captured in mouth of 5 nm nanopore

•Negatively charged DNA is attracted to Negatively charged DNA is attracted to electrostatic potential of silicon electrostatic potential of silicon nanoporenanopore

DNA threaded through the DNA threaded through the nanopore using a salt gradientnanopore using a salt gradient

Why a Salt Gradient?

•Salt creates more Salt creates more electrostatic potentialelectrostatic potential

•Increased capture by Increased capture by 30 fold30 fold

•Were able to capture Were able to capture & detect DNA at & detect DNA at picomolar picomolar concentrationsconcentrations

Now…what about detecting bases?

• “Electronic Signatures of all four DNA nucleosides in a Tunneling Gap” Nano Letters. 2010

• Arizona State University- lead by Stuart Lindsay

• Tunneled single dNTPs through a 2.5 nm gap

• Used electronic signals detected with a gold plated probe to “read” nitrogenous bases

Electron tunneling & base detection

• DNA forced through small 2.5 nm gap

• Captured between gold plate and probe charged with 4-mercaptobenzoic acid

• Electronic signal measured– Different bases release

difference signal strengths

– Hydrogen bonding

Electronic signatures & configurations

Data Output

Different peaks for individual base Different peaks for individual base detection with acid additiondetection with acid addition

Electrodes with no acid have no Electrodes with no acid have no separationseparation

Nanotechnology…Conclusions?

• Much more cost effective

• Still considerable amount of research to be completed

• High potential– Sequence a full human genome in 1 day!

Links & ResourcesJournal article citations:• Wanunu et al.“Electrostatic focusing of unlabelled DNA into nanoscale

pores using a salt gradient.” Nature Nanotechnology. 20 December 2009

• Chang et al. “Electronic Signatures of all Four DNA Nucleosides in a Tunneling Gap.” Nano Letters, 2010

Links to News Articles:Lindsay’s Universal DNA Reader- from Science Daily & Nanotechnology Now

http://www.sciencedaily.com/releases/2010/02/100211175212.htm

http://www.nanotech-now.com/news.cgi?story_id=36735Boston University’s DNA threader

http://www.eurekalert.org/pub_releases/2009-12/buco-bur121809.php

Pacific Biosciences SMRT Website:

http://www.pacificbiosciences.com/index.php?q=technology-introduction