Nanofluidic-enabled single-molecule

imaging for DNA analysis

 Single Molecule DNA Imaging

• Objectives
• Handle wide range of DNA sizes, PCR to whole
chromosome
• Enable sequence or structure-specific single-molecule
interrogation
• Strategy
• Linearize DNA for native-state structural analysis
• Massively parallel processing for rapid reduction to
single molecule
• Simple sample handling and image capture
Genomic DNA Analysis Challenge
 Step 1: DNA Linearization
Image of single DNA fragments
in a BNM chip

Image of millions of DNA

fragments in gel

DNA linearization requires True high-resolution No other DNA

a nano-fluidic platform single-molecule elongation strategy
analysis becomes enables full analysis
possible
Step 2: Parallel Processing

Challenge: sort macro or microfluidic

sample out to enable single molecule
sorting and analysis with high
processivity and high resolution
 High Density Format

1.3 cm

Lambda 50kb ~20um

BAC clone
170kb ~60um

Up to ½
kilometer of
channels per
square
centimeter
Step 3: Simple System Format
NanoAnalyzerTM

Fluorescent microscope
stage and optics,
nanofluidic chip, reagents
DNA Linearization

~1,000,000 bp
~1000 channels
High Molecular Weight Unwinding
DNA Integrity Within Chip
 Size Measure Matches Standard
No size bias in DNA movement into chip
20-250 molecules per size class counted
Sequence Labeling

Raw Data is “bead on string”

 Mapping In Solution
12.7kb ~5kb 13kb 10.5kb 8kb

Lambda Genomic
DNA Restriction
Mapping

Long Range PCR

Product Haplotyping
 DNA Barcoding Examples

Human BAC clone 176,000 bp

Lambda DNA

Long range PCR product

No TIRF
Single fluor detection sensitivity
NCI collaboration Genotoxicity
 NCI Collaboration Genotoxicity

Size analysis generated for

Image recognition the sample population
software counts and
measures DNA fragments Average DNA 9
microns in length,
approximately 54 kbp

Largest DNA
fragment in
Image Processing sample:
288 kbp
CGI Collaboration DNA Sequencing

Targets:
1 billion raw bases per minute
> Gold standard accuracy
$100 materials costs per genome
 NanoChip Applications

• Nanofabrication:
Enable single molecule
• Reduce sample size patterning and analysis
– Single cell
by imaging
• High mol. Wt. Single
molecule
– 10kb - >1M kb

Haplotyping

Genotox
DNA/Protein
Sequencing
 Team

Han Cao, PhD CTO,

Founder
Ming Xiao, PhD
Mike Austin, PhD
Paru Deshpande, PhD
Swamy Vijayan, PhD
Nisha Fernandez, MS

Funding: NHGRI 5R43HG004199-02 SBIR phase 1

NCI 5R43CA120611-02 SBIR phase 1

mbj@bionanomatrix.com