Download A Novel Biocompatible Monolithic Polymer for Use in Nucleic Acid

ANovelBiocompatibleMonolithicPolymerforUseinNucleicAcidExtraction,DNAPurificationandDNASequencing.
MarkDobbs,IvanRueda,SuzyKwon,VictorShumandKeithAOberg
MonolythixInc.CamarilloCA
With increasing demand for molecular genetic testing comes an increased demand for non-invasive cell sampling technologies for nucleic acid extraction. We describe a novel
hydrophilic polymer monolith that has been used to robustly extract nucleic acids from human buccal epithelial cells. The methods and material described here result in high
efficiency extraction of good quality genomic DNA from single buccal swabs. With a normal cell count, the process extracts > 800ng of high molecular weight genomic DNA from a
single swab. In addition, where higher concentrations of DNA are required, we describe a method for pooling and concentrating the DNA from multiple swabs using the monolith
in a novel format. The monolithic polymer can also be used to purify PCR products for use in downstream reactions, and to purify sequencing reaction products. We present data
from sequential processing of DNA from buccal cells to sequencing results where all purification steps are mediated by the monolith polymer.
G
H
7
8
9
10
11
12
23.7 NoCq 22.8 NoCq 22.5 NoCq 22.2 NoCq 23.4 NoCq 23.3
Std.
NoCq 23.1 NoCq 23.1 NoCq 22.7 NoCq 22.6 NoCq 23.3 NoCq 23.5
23.1 NoCq 23.5 NoCq 22.9 NoCq 22.6 NoCq 23.2 NoCq 23.3 NoCq
NoCq 23.2 NoCq 23.0 NoCq 22.6 NoCq 22.6 NoCq 22.5 NoCq 23.5
23.1 NoCq 23.4 NoCq 22.4 NoCq 22.6 NoCq 23.5 NoCq 23.2 NoCq
NoCq 22.7 NoCq 23.0
Sequencing
Products
Std.
22.6 NoCq 22.3 NoCq 23.3 NoCq 23.4
2000
1.89
1.92
1767
1732
1.86
1628
2.00
1639
Elution1
1500
1.50
1.00
1000
0.50
500
93
0
0.00
P1
P6
P11
P16
PosCtrl
PlateNumber
NegCtrl
n=20wellsperplate
Buccal cell extraction performance
Elution2
characteristics. Elution
1 has > 80% of
DNA (> 1000 ng in 60 uL, >16 ng/uL).
Second 60 uL elution adds about 20%
more DNA.
Buccal cell DNA Integrity. Elution 1 DNA
has a strong HMW region with minimal
smearing/degradation. Elution 2 has
equivalent integrity, but is lower in
concentration (not shown).
52.7ng/uL
Concentration Method:
1.
2.
3.
4.
845 uL dilute buccal DNA (17.5 ng/uL)
Add 210 uL 1M MOPS, pH 6.8 plus 1.0 mL of DDW
Split into 4 parts, apply to 4 monoliths in P1000 tips
Perform two sequential 30 uL elutions, pool second
elutions
ModelPointofCareDiagnosticDeviceinaP1000Tip
WickintoTip
Mix
WholeBlood
Dropof
Blood
into
Isotonic
Buffer
Detector
Wick
up
1min
StainedWhiteCellsTrappedatTip
DetectionofCFTRgeneinWhiteCells:
AmplificationatendofMonolithTip
LightSource
1530
2.08
Successful amplification, PCR
product purification, and Sanger
product purification was achieved
for 4 targets ranging in size from
180bp to 5100bp
Trackingmovementofbloodcellsinmonolith
Saliva
Cells
(~80%
WBC)
1.93
• Purifyusing
MLXPurifier
(15-30min)
• Sanger
Sequencing
Cap.GelRun
• High
Concentration
Applications,
e.g.NGS
Stainwith
MethyleneBlue
andRinse
BuccalKitDNAYield
23.0 NoCq 22.8 NoCq 22.4 NoCq 21.9 NoCq 23.1 NoCq 23.0
16 replicate Buccal Cell Extractions of 3 different samples
in a checkerboard array. For 3 of 48 negative control
wells, a quantitative standard DNA was used in the real
time reaction (Std.) The negative controls all had no real
time PCR amplification curve. Mean percent recoveries
based on input cell counts were from 82% to 99%.
• Purifyusing
MLXPurifier
(~15min)
• Prepare
Sequencing
Reactions
PCR
Products
6
Std.
F
BuccalCells
5
B
E
• ExtractDNA
usingMLXBCGEKit
(~1hr)
• AmplifyPCR
Fragments
(different
lengths)
4
22.9 NoCq 22.9 NoCq 22.6 NoCq 22.7 NoCq 23.0 NoCq 22.9 NoCq
D
BuccalCellExtractionMonolithin96WellFormat.
3
A
C
2
A260/A280
1
DNAYi eld(ng)
RelativeSignalIntensity
0.3min
LightSource
10-40min
Detector
Positive
620
PlusPolymeraseTestSample
2.0min
DetectionofDNAEgeneafterwickingofWholeBlood
spikedwithE.coli:AmplificationInsideMonolith
180
NoPolymeraseControl
Transfer
Tipto
LAMP
Mix,Heat
Transfer
Tipto
Chase
Solution
HorizontalMonolithCone(Split)
VerticalMonolithCone
Negative
SignalofPositivesamplewas
stronginternally
DetectionofDNAEgeneafterwickingofBacteria:
LAMPReagentsDriedinMonolith
LightSource
Detector
Positive
HorizontalMonolithCone(Split)
Negative
SignalofPositivesamplewas
stronginternally
Model of a self-wicking point of care device. Raw sample
can be loaded at left, filtered, extracted, and amplified in
sequential processing zones. Detection would occur in
restricted zone after all processing is done. The sink
(right) ensures adequate and continuous fluid flow.
DigestionofBSAusingimmobilizedProteinaseK.BSAsolution
waswickedintoProteaseZone,heldfor15minutesandwicked
intotheCollectionZonebeforeanalysisbyHPLC.
IntegratedDeviceMilestones
1. ExtractionofpuregDNA frombuccalcells
2. Bound,(concentrated),andreleasedpurebuccalcellDNA
3. Separationofwhitebloodcellsandbacteriafromredbloodcellsbywicking
4. Insituisothermalamplificationoftargetspecificgenesinmonolith(LAMPon
WBC,E.coli,Flu)
5. InsitudetectionofamplificationusingaDNAbindingdye
6. ImmobilizationofactiveproteinaseK(andlysozyme)inmonolith
7. PreservationofLAMPreagents,includingpolymerase,inmonolith