DRAGEN

(Direct Rapid Analysis Generating Extracted Nucleotides)

David Ross, Elizabeth Strychalski, Erica 

Butts, Pete Vallone (NIST)

Alyssa Henry, Chris Konek (Applied 

Research Associates, Inc.)

Funding From:

US Army Criminal Investigation Lab

Army Research Laboratory

NIST, OLES
Gradient Elution Moving Boundary 

Electrophoresis (GEMBE)

• Simple, Robust System

• High Quality Data

• Minimal Sample Prep 

Requirements

• Applied to the 

detection of DoDrelevant species from 

crude matrices
How GEMBE Works

Simple microfluidic system that provides multi-step 

chemical analysis functionality

Sample Buffer

Capillary
How GEMBE Works

Controlled counterflow prevents proteins, enzymes 

and particulates from entering and fouling the 

capillary

Proteins ATP Cl DNA- K+

Enzymes

Particulates

High 

Voltage

electrophoresis

counterflow
Kit-Based Platform

Fission product detection Nerve agent detection Explosives detection

Bioagent detection

DNA Extraction?
Gradient Elution Isotachophoresis (GEITP)

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Run Buffer (Fast Ions)

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Sample Buffer

(Slow Ions)

Capillary

Capillary
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GEITP Theory

Fluid flow

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Electrophoresis

Run Buffer (Fast Ions)

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DNA

Capillary

Capillary
How It Works: GEITP Extraction of 

Human DNA

movie
Phase I Project Goals:

• Extract DNA from Crude 

Samples

– High copy number 

samples, but with PCR 

inhibitors

– ‘Dirty Buccal Swab’

• Quantitate DNA

– On-line fluorescence 

detection

• Deliver Clean DNA 

suitable for STR typing

– Small final volume (10 μL)

• Format potentially 

suitable for field use

Cell lysis

DNA Extraction

Delivery
Dirty Buccal Swab

• Buccal Swab plus 50-

100 mg soil

– Soil obtained from 

under oak tree grove 

at NIST

• 500 mL lysis buffer 

solution

– 485 mL trailing 

electrolyte (with 

Triton-X 100)

– 15 mL Proteinase K 

• Incubate at 56 °C for 

15 min
yp

formandmethodallowed rapidextraction, purification, concentrat

on, anddeliveryof DNAusingGEITP.

GEITP Hardware

• Leading Electrolyte 

Reservoir

• Sample

• Capillary

Leading

Electrolyte

Sample

Capillary
yp

formandmethodallowed rapidextraction, purification, concentrat

on, anddeliveryof DNAusingGEITP.

GEITP Hardware

• Leading Electrolyte 

Reservoir

• Sample

• Capillary

• High Voltage

• Pressure Controller

• Detectors

– Fluorescence (LIF)

• SYBR Green Dye

– Conductivity (C4D)

HV Leading

Electrolyte

Sample

Pressure

Controller

laser in

(488 nm)

C4D
GEITP Extraction Method

DNAdeliveryfromcrudesamples

• Our platform and method allo

wed rapid extraction, purification, concentration,

quantification, and deliveryof DNA using GEITP.

• Delivered D

NAwas of sufficient amount and purity for qPCR and multiplex

PCR for human identification using STR typing.

“Human identification from crude samples” E.A. Strychalski, C. Konek, E.L.R. Butts, P.M.Vallone,A.C. Henry, D. Ross. In Preparation, 2012.

a) Mix buccal swab, dirt, and lysis buffer;

incubate (15 min, 56 °C)
GEITP Extraction Method

DNA delivery from crude samples

• Our platform and method allowed rapid extraction, purification, concentration,

quantification, and delivery of DNA using GEITP.

• Delivered DNA was of sufficient amount and purity for qPCR and multiplex

PCR for human identification using STR typing.

“Human identification from crude samples” E.A. Strychalski, C. Konek, E.L.R. Butts, P.M.Vallone,A.C. Henry, D. Ross. In Preparation, 2012.

b) Dip capillary into sample; start with positive 

pressure
GEITP Extraction Method

DNA delivery from crude samples

• Our platform and method allowed rapid extraction, purification, concentration,

quantification, and delivery of DNA using GEITP.

• Delivered DNA was of sufficient amount and purity for qPCR and multiplex

PCR for human identification using STR typing.

“Human identification from crude samples” E.A. Strychalski, C. Konek, E.L.R. Butts, P.M.Vallone,A.C. Henry, D. Ross. In Preparation, 2012.

c) Turn on High Voltage (2000 V); reduce pressure;

focus/extract DNA for 1 minute
GEITP Extraction Method

DNA delivery from crude samples

• Our platform and method allowed rapid extraction, purification, concentration,

quantification, and delivery of DNA using GEITP.

• Delivered DNA was of sufficient amount and purity for qPCR and multiplex

PCR for human identification using STR typing.

“Human identification from crude samples” E.A. Strychalski, C. Konek, E.L.R. Butts, P.M.Vallone,A.C. Henry, D. Ross. In Preparation, 2012.

d) Reduce pressure to move DNA through conductivity 

and LIF detectors
GEITP Extraction Method

DNA delivery from crude samples

• Our platform and method allowed rapid extraction, purification, concentration,

quantification, and delivery of DNA using GEITP.

• Delivered DNA was of sufficient amount and purity for qPCR and multiplex

PCR for human identification using STR typing.

“Human identification from crude samples” E.A. Strychalski, C. Konek, E.L.R. Butts, P.M.Vallone,A.C. Henry, D. Ross. In Preparation, 2012.

e) Turn off High Voltage; move focused DNA past LIF 

detector at controlled rate; quantitate DNA
GEITP Extraction Method

DNA delivery from crude samples

• Our platform and method allowed rapid extraction, purification, concentration,

quantification, and delivery of DNA using GEITP.

• Delivered DNA was of sufficient amount and purity for qPCR and multiplex

PCR for human identification using STR typing.

“Human identification from crude samples” E.A. Strychalski, C. Konek, E.L.R. Butts, P.M.Vallone,A.C. Henry, D. Ross. In Preparation, 2012.

f) Deliver DNA to clean sample tube
DNA Quantitation

DNA quantification

egions indicate data used to calculate the LIF peak area for DNA 

on. qPCR provides another measure of the amount of delivered DNA.

mega standard

Buccal swab

ed buccal swab

Standard DNA

(Human) 

0.5 ng/μL

Clean Buccal

Swab

Dirty Buccal

Swab

Ct = 30.7

0.024 ng/μL

Ct = 29.8

0.216 ng/μL

Ct = 30.2

0.157 ng/μL
DNA Quantitation

DNA quantification

• By calibrating the optical measurements against qPCR measurements in this way,

it is possible to estimate the amount of DNA delivered during analysis in real time.

Promega standard

Soiled buccal swab

Soiled swab without

buccal sample

Buccal swab

“Human identification from crude samples” E.A. Strychalski, C. Konek, E.L.R. Butts, P.M.Vallone,A.C. Henry, D. Ross. In Preparation, 2012.

needs 

Data from dirty buccal swabs have larger LIF signals than data from clean samples, for a 

given qPCR measurement, due to non-human DNA in the soil.

Dirty

Dirty
Human Identification

R

Length (Basepairs)

elative fluorescence units

Buccal swab Soiled buccal swab

• Comparable STR profiles were obtained from DNA delivered from buccal swabs

and soiled buccal swabs from the same person.

Human I dentification

“Human identification from crude samples” E.A. Strychalski, C. Konek, E.L.R. Butts, P.M.Vallone,A.C. Henry, D. Ross. In Preparation, 2012.

Length (Basepairs)

• Delivered DNA was of sufficient amount and purity for qPCR, multiplex PCR, and

STR typing for human identification from buccal swabs and soiled buccal swabs.

Clean Buccal Swab Dirty Buccal Swab

Comparable STR profiles were obtained from DNA delivered from both clean and dirty 

buccal swabs from the same person.

Delivered DNA was of sufficient amount and purity for qPCR, multiplex PCR, and STR 

typing for human identification from both clean and dirty buccal swabs.
Dirty Swab

Lth (Bi)

Soiled buccal swab
Human Identification

Sample Concentration 

in sample Loci Called n

Concentration 

Delivered

DNA Standard 0.5 ng/μL -mixture- 2 .037 ng/μL

Blank Swab - 0/16 1 -

Blank Dirty 

Swab - 0/16 1 -

Clean Buccal 

Swabs

1 x (16 ± 0)/16 6 0.136 ng/μL

1/3 x (14 ± 2)/16 6 0.057 ng/μL

1/10 x (3 ± 4)/16 6 0.017 ng/μL

Dirty Buccal 

Swabs

1 x (16 ± 0)/16 6 0.174 ng/μL

1/3 x (15 ± 3)/16 6 0.097 ng/μL

1/10 x (10 ± 4)/16 6 0.031 ng/μL

1x concentrations of both clean and dirty buccal swabs gave STR profiles with 16/16 

loci called for every sample.
Conclusions

• DRAGEN-based DNA extraction and 

quantitation

– Time required < 5 minutes 

– DNA concentration and purity good enough for 

human ID

• High copy number sample
What Next?

• Improve Extraction Efficiency

– Currently ~1%

– Conventional techniques >20%

• Demonstrate with other sample types

– Bone

– Chewing Gum

– Trace Samples

– Plant Material
Acknowledgements

Funding From:

US Army Criminal Investigation Lab

Army Research Laboratory

NIST, OLES

This material is based upon work supported by the US Army Contracting

Command - Army Proving Ground under Contract No. W911QX-11-C-0089-P00002.

The findings in the report are not to be construed as an official Department

of Army position unless so designated by other authorized documents.

Citations of manufacturer's or trade names does not constitute an official

endorsement or approval of the use thereof.