Nanofluidic Enabled Single Molecule Imaging for DNA Analysis
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Transcript of Nanofluidic Enabled Single Molecule Imaging for DNA Analysis
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Nanofluidic-enabled single-molecule
imaging for DNA analysis
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Single Molecule DNA Imaging
Objectives Handle wide range of DNA sizes, PCR to whole
chromosome
Enable sequence or structure-specific single-moleculeinterrogation
Strategy Linearize DNA for native-state structural analysis
Massively parallel processing for rapid reduction tosingle molecule
Simple sample handling and image capture
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Genomic DNA Analysis Challenge
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DNA linearization requiresa nano-fluidic platform True high-resolutionsingle-moleculeanalysis becomespossible
Image of single DNA fragmentsin a BNM chip
Image of millions of DNAfragments in gel
No other DNAelongation strategyenables full analysis
Step 1: DNA Linearization
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Step 2: Parallel Processing
Challenge: sort macro or microfluidic
sample out to enable single molecule
sorting and analysis with high
processivity and high resolution
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1.3 cm
High Density Format
Up to
kilometer of
channels persquare
centimeter
Lambda 50kb ~20um
BAC clone
170kb ~60um
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NanoAnalyzerTM
Fluorescent microscopestage and optics,nanofluidic chip, reagents
Step 3: Simple System Format
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~1000 channels
DNA Linearization
~1,0
00,0
00b
p
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High Molecular Weight Unwinding
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DNA Integrity Within Chip
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Size Measure Matches Standard
No size bias in DNA movement into chip
20-250 molecules per size class counted
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Sequence Labeling
Raw Data is bead on string
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Mapping In Solution
12.7kb ~5kb 13kb 10.5kb 8kb
Lambda Genomic
DNA Restriction
Mapping
Long Range PCR
Product Haplotyping
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DNA Barcoding Examples
Long range PCR product
Lambda DNAHuman BAC clone 176,000 bp
No TIRF
Single fluor detection sensitivity
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NCI collaboration Genotoxicity
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Image recognitionsoftware counts andmeasures DNA fragments
Size analysis generated for
the sample population
Average DNA 9microns in length,approximately 54 kbpLargest DNAfragment insample:288 kbp
NCI Collaboration Genotoxicity
Image Processing
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Targets:1 billion raw bases per minute
> Gold standard accuracy
$100 materials costs per genome
CGI Collaboration DNA Sequencing
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NanoChip Applications
Nanofabrication:
Reduce sample size
Single cell
High mol. Wt. Single
molecule
10kb - >1M kb
Genotox
Haplotyping
SequencingDNA/Protein
Enable single molecule
patterning and analysis
by imaging
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Team
Funding: NHGRI 5R43HG004199-02 SBIR phase 1NCI 5R43CA120611-02 SBIR phase [email protected]
Han Cao, PhD CTO,
Founder
Ming Xiao, PhD
Mike Austin, PhD
Paru Deshpande, PhDSwamy Vijayan, PhD
Nisha Fernandez, MS