PURIGEN BIOSYSTEMS Ionic Purification SystemIsotachophoresis (ITP) separates and concentrates charged molecules in solution solely based on their electrophoretic mobility. Biological

© 2019 Purigen Biosystems, Inc. All rights reserved.

Ionic™ Purification SystemSimplified Nucleic Acid Purification

BROCHUREVERSION 1.1 | OCTOBER 2019

Purigen Biosystems, Inc.5700 Stoneridge Drive, Suite 100

Pleasanton, CA 94588

TEL: +1 925 264-1364 EMAIL: [email protected]

PURIGEN BIOSYSTEMS

https://www.purigenbio.com


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BROCHURE | IONIC™ PURIFICATION SYSTEM

The commonly used bead- and column-based extraction technologies have followed the same fundamental workflow for over 20 years. This workflow uses ethanol, chaotropic salts, and other solutions to bind nucleic acid to a silica membrane or surface-labeled bead, which is then washed prior to the nucleic acid being stripped off the solid support into an elution buffer. During this typically laborious process, the nucleic acid is denatured, dehydrated, and fragmented. The eluate is also susceptible to contamination from wash buffers or beads.

Why We Developed a New Approach to Nucleic Acid Purification

Disadvantages Consequences

Incomplete binding to or removal from the solid support • Nucleic acid loss compromises data quality when sample input quantity is limited

• Recovery can be biased by fragment length or GC content

• For researchers, reduced biological insight

• For clinicians, less actionable information and false negatives

Contamination from wash buffers and bead coatings • Low purity leading to false negatives and compromised data

Workflow with multiple hands-on steps • Throughput bottleneck and potential errors; excessive use of disposable tips and labware

Process and Characteristics

• Surface-based = loss and damage

• Wash solution contamination

• Limited integration potential

Traditional Purification Separation Principle

Column-based Silica

Bead-based Carboxyl

Solubility


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Impurity

Trailing Ions (slow) Leading Ions (fast)

Nucleic Acid

1 Sample loaded

CathodicBuffer

SeparationBuffer

ElutionBuffer

AnodicBuffer

Nucleic Acid

2 Current applied; nucleic acid moves and concentrates

CathodicBuffer

SeparationBuffer

ElutionBuffer

AnodicBuffer

Nucleic Acid

3 Purified sample collectedNucleic Acid

Impurities Nucleic Acid

Purigen Biosystems

Isotachophoresis (ITP) separates and concentrates charged molecules in solution solely based on their electrophoretic mobility. Biological samples are gently lysed and added to the Purigen Ionic™ Fluidics Chip. An electric field is then applied to the chip and the nucleic acid is isolated in its natural, native form. The nucleic acid is not denatured or dehydrated, and there’s no binding to, or stripping from, fixed surfaces. The result is a higher yield of pure nucleic acid that is less fragmented and free from bead or wash buffer contamination.

Isotachophoresis – a Superior Approach to Nucleic Acid Separation

Process and Characteristics

• Solution-based = higher yield and integrity

• Higher purity

• Fewer processing steps and less hands-on time

Purigen ITP Separation Principle

ITP Charge

Simple Charge-based Sample Prep in Solution

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Ionic™ Purification System

The revolutionary Ionic™ Purification System from Purigen Biosystems utilizes isotachophoresis (ITP) to extract, purify, and concentrate nucleic acid from biological samples. Nucleic acids extracted by the system are pure, abundant, and ready to use in just 60 minutes.

➊ Touchscreen console; Software-driven workflow

2 Thermally controlled chip holder

3 Compact benchtop footprint

➊

3

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Simplified Nucleic Acid Preparation

The Ionic system is so different, its advantages are most readily understood in contrast with conventional nucleic acid extraction and purification methods:

No organic solvents

No harsh, high-salt buffers

No system programming

No beads or repetitive washing

No hands-on mixing, separation, sample transfers, or buffer exchanges

No pumps, valves, or other moving parts


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Purigen Biosystems

Ionic™ Fluidics Chips

Biological samples are loaded into the 8 sample wells of the Ionic fluidics chip. Nucleic acid has higher electrophoretic mobility relative to unwanted lysate components. By applying an electric field across the length of a chip microchannel, the Ionic system separates and concentrates nucleic acid between buffers with higher and lower mobilities. Impurities fall behind the low mobility buffer and are separated from target nucleic acids. As the target nucleic acids pass through the channel, an integrated sensor stops the current once nucleic acids reach the elution well.

INSTRUMENT SPECIFICATIONS

Dimensions and Weight (includes leveling platform)

Height 44 cm / 17.3 in

Width 45 cm / 17.7 in

Depth 52 cm / 20.5 in

Weight 24.5 kg / 54 lb

Facility Requirements

Power 110-120 VAC, 60Hz, 4A

Regulatory Compliance

UL 61010

FCC CFR title 47, sub part B of part 15 / ICES-003, Issue 6

CHIP SPECIFICATIONS

Dimensions

Height 14.53 mm / 0.57 in

Width 85.48 mm / 3.37 in

Depth 127.76 mm / 5.03 in

Working Volumes

Sample well volume 200 µL (typical)

Buffer well volume Varies by protocol, sample type

Elution well volume 40 µL (typical)

Storage Conditions

Temperature 15 – 32⁰C

Packaging In sealed, static dissipative, bag

Handling With disposable gloves, avoiding contact with surface

Orienting Feature

Removable Protective Sealon sample wells

Tracking Label

Elution Wells

Sample WellsBuffer Wells

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Rapid Purification of Precious Samples in Just ONE Hour

Ionic System Workflow

60 minTotal Run-time

< 3 minutes total hands-on time per sample

2Load SamplesLoad 8 samples – use touchscreen to start the run

50 mins

Load Run BuffersAdd buffers from reagent kit – use touchscreen to start priming

8 mins1

Get Purified Nucleic AcidCollect samples when touchscreen displays “Run Complete”

2 mins3


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Purigen Biosystems

Higher Quality Nucleic Acid for Higher Quality Data

The Ionic system produces higher quality nucleic acid due to less fragmentation and no risk of contamination from wash buffers or beads. When higher quality material is input into downstream analysis, superior results can be obtained; providing more biological insight for researchers or more robust and reliable results for clinicians.

FIGURE 2: Cell populations of 56K, 11.2K and 1.12K were purified using Purigen and an automated column-based approach (each cell population was run in replicates of 6 for each technology). The purified samples were then run in a qPCR reaction with a 280bp target.

• The 11,200 cell samples purified using Purigen demonstrate a threshold cycle of ~3 lower than the cells purified by a column-based approach. This equates to close to a 8-fold increase in amplifiable yield when using the Purigen technology.

• The 1,120 cell samples purified using Purigen are all clearly detected, however 3 of the column purified samples do not contain detectable amounts of DNA. The resulting data interpretation for the column-based samples would likely be a positive detection with a very low confidence level, or the assignment of a negative result (and potentially a recommendation for a re-test).

Purigen Cells to Pure DNA Kits Provide Superior Quantitation / Detectionvs. Automated Column-based Kits

Purigen-purified cell samples demonstrate:

Higher purity DNA with less fragmentation and no contamination

Improved amplifiable yields ~8x

FIGURE 1: 12 cell samples purified using Purigen and a conventional column-based approach (replicates of 6 for each technology). Purigen-purified samples show clean target bands; without smearing and less fragmentation.

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Purigen Cells to Pure DNA Kit vs. Automated Column-based Kits

gDNA Extracted from 56,000 Cells

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Purigen Cells-to-DNA Provides Higher Quality DNAvs. Automated Column-based System

Purigen Automated Column-based System

(gDNA extracted from 56,000 cells)

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PURIGEN Automated Column-based Kits

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15




Replicates Replicates

40

35

30

25

CT V

alue

s (lo

g 2)Lo

wer

is b

ette

r

PURIGEN

Automated Column-based Kit

Advantage of approximately ~3 CTs

qPCR Data for ~280 bp AmpliconElutes exceeded cycle threshold

11,200 Cells56,000 Cells 1,120 Cells

Load 8 samples – use touchscreen to start the run

Add buffers from reagent kit – use touchscreen to start priming

Collect samples when touchscreen displays “Run Complete”

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Superior Nucleic Acid Recoveries from FFPE Samples

The Ionic system produces more DNA from FFPE samples without requiring the use of slides (slide use and paraffin removal is optional). The ability to obtain comparable nucleic acid yields when using scrolls (versus slide mounted FFPE slices) greatly simplifies the workflow when sample micro-dissection is not required. This allows projects to be completed faster and at a lower cost.

Purigen’s FFPE to Pure DNA protocol greatly simplifies the processing of FFPE samples. The hands-on time per sample is less than three minutes and enables working directly from scrolls. The protocol also eliminates the need for paraffin removal.

Simplified FFPE Workflow Saves Time and Money

Column-based Kit WORKFLOW

1Sample*

5Binding

2Remove Paraffin

6AW1

Wash

3Lyse

7AW2 Wash

4Heat

8EtOH Wash

9Elute

10Spin Dry

11Ready- to-use

DNA

*Columns can typically yield only ~15% of the DNA from scrolls as compared to slides

Purigen WORKFLOW

*Compatible with scrolls or slides

Processed in batches of 24

2Lyse

1Sample*

No need to trim wax or remove

paraffin

5Ready- to-use

DNA

3Heat

4Purify

Ionic™ Purification

SystemSimple hands-off thermo-mixer

protocol provided

Lysis Purification

< 3 minutes of hands-on time per sample


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Nucleic acid yields from scrolls demonstrate an increased median yield of 31x when using the Ionic Purification System vs. a column-based kit.

Purigen Biosystems

FIGURE 4: Results for 32 FFPE block samples* purified from scrolls for Purigen (no paraffin removal) vs. slides (excess paraffin removed) for a column-based approach. Yields for Purigen samples demonstrate improved amplifiable yields of on average 3.5x versus column-based approach.

qBio

Am

plifi

able

Yie

ldPu

rigen

/ Co

lum

n Ki

t 101

100

qBio

Rat

io S

lide

BF BG BH BI BJ BK BL BM BN CL CM CN CO CQ CR CS LI LJ LK LM LN LO LP LQ LRCP

10-fold

Parity

Block

BREAST COLON LUNG

Selected for Sequencing

* All blocks were supplied by Discovery Sciences with the age of storage ranging from 3 to 24 years. Comparisons were preformed using interleaved scrolls from each FFPE block - scrolls were slide mounted before nucleic acid extraction using a column-based kit.

80 bp qPCR Assay

Purigen SCROLLS vs. Column-based Kit SLIDES

Nucleic acid yields from scrolls using the Ionic Purification System are on average 3.5x higher when compared to yields from slide-mounted slices using a column-based kit.

FIGURE 3: Results for 32 FFPE block samples* purified from scrolls for both Purigen and for a column-based approach. Yields for Purigen samples demonstrate improved amplifiable yields with a median increase of 31x versus a column-based approach.

qBio

Am

plifi

able

Yie

ldPu

rigen

/ Co

lum

n Ki

t

100

qBio

Rat

io S

crol

l

BD BE BH BI BJ BK BL BM BN CL CM CN CO CP CQ CR CS LI LJ LK LM LN LO LP LQ LR

Block

BREAST COLON LUNG

10-fold101

102

103

104BF BG CJ CK LG LH

100-fold

1000-fold

* All blocks were supplied by Discovery Sciences with the age of storage ranging from 3 to 24 years. Comparisons were preformed using interleaved scrolls from each FFPE block.

80 bp qPCR AssayRatio of Amplifiable Yield

Ratio of Amplifiable Yield

Purigen SCROLLS vs. Column-based Kit SCROLLS

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In addition to increased yields and a greatly simplified workflow, data quality is also improved. Data below shows purified DNA from FFPE samples analyzed using the Agilent SureMASTR Tumor Hotspot sequencing panel which includes 252 amplicons ranging in size from 128–245 bps. To highlight coverage differences related to the sample purification technology, all data

was normalized to a reference sample to remove the effect of coverage differences introduced by differences in target amplicon amplification efficiencies. The reference data set was generated using the average results obtained from the purification of a high-quality sample using both Purigen and column-based techniques (as such the reference sample is not biased to either technology).

Improved Data Quality from the Ionic Purification System

More Uniform Coverage (when compared to column-based kits)

Chromosome Number

0.5

0.0

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-1.0

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-2.0

-2.5

-3.0

chr1

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chr1

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chr1

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chr1

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chr2

chr3

chr4

chr5

chr6

chr7

chr8

chr9

PURIGEN

Column-based Kit

FIGURE 5: Results shown on a chromosome-level. Ionic system eluates show tighter clustering more centered around the zero line. This is indicative of more uniform sequencing coverage.

Ionic system purification shows no bias towards amplicon lengthIonic system purification shows no bias towards GC content

FIGURE 6: Results shown relative to amplicon length and amplicon GC content. Purigen-purified samples show superior uniformity for both amplicon length and GC content, with very little deviation from the expected coverage. Column-based purification shows a bias towards shorter amplicon lengths and lower GC content amplicons.

0 40 60 80 100 120 140 160

0.5

0.0

-0.5

-1.0

-1.5

-2.0

-2.5

-3.0

30 40 50 60 70 80

0.5

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-2.5

-3.0

Log

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ence

0 40 60 80 100 120 140 160

Length GC ContentLength GC Content30 40 50 60 70 80

Coverage Ratio vs. Length Coverage Ratio vs. % GC Content

PURIGEN Column-based Kit PURIGEN Column-based Kit

Ionic System Produces Less Coverage Bias

Log

Rat

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ampl

e/R

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No Bias Toward Length or GC Content of Amplicons

FIGURES 7 and 8: Data points highlighted in teal indicate that “tailing” data points in column-based purifications seem to be a result of bias against longer length amplicons. Data points highlighted in maroon indicate that “downward shifts” of chromosome specific data seem to be a result of bias against longer amplicons and higher GC content amplicons.

Chromosome Number

0.5

0.0

-0.5

-1.0

-1.5

-2.0

-2.5

-3.0

chr1

chr1

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chr1

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chr1

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chr1

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chr1

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chr1

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chr1

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chr1

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chr1

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chr1

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chr2

chr3

chr4

chr5

chr6

chr7

chr8

chr9

Purigen-purified FFPE samples demonstrate:

3.5x higher yield on average from scrolls or slides

No bias toward length or GC content

More uniform sequence coverage

Length

Coverage Ratio vs. Length

Length

Coverage Ratio vs. Length

PURIGEN

0.5

0.0

-0.5

-1.0

-1.5

-2.0

-2.5

-3.0

20 40 60 80 100 120 140 160

uniform coverage at all lengths

GC Content

Coverage Ratio vs. % GC Content

Column-based Kit

0.5

0.0

-0.5

-1.0

-1.5

-2.0

-2.5

-3.0

30 40 50 60 70 80

lower coverage at higher GC content

Purigen Biosystems

GC Content

Coverage Ratio vs. % GC Content

PURIGEN

0.5

0.0

-0.5

-1.0

-1.5

-2.0

-2.5

-3.0

30 40 50 60 70 80

uniform coverage for all GC content

Column-based Kit

0.5

0.0

-0.5

-1.0

-1.5

-2.0

-2.5

-3.0

20 40 60 80 100 120 140 160

lower coverage at longer length

Log

Rat

io S

ampl

e/R

efer

ence

Log

Rat

io S

ampl

e/R

efer

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Log

Rat

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ampl

e/R

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Purigen Biosystems, Inc.5700 Stoneridge Drive, Suite 100Pleasanton, CA 94588

TEL: +1 925 264-1364 EMAIL: [email protected]

© 2019 Purigen Biosystems, Inc. All rights reserved.Ionic™ is a trademark of Purigen Biosystems, Inc., in the U.S. PUR-DOC-10


Documents

PURIGEN BIOSYSTEMS Ionic Purification SystemIsotachophoresis (ITP) separates and concentrates charged molecules in solution solely based on their electrophoretic mobility. Biological