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Rapid Sample Lysis in a Miniaturized Format
Bruce Irvine
Chief Technology Officer
Claremont BioSolutions LLC
Sample Prep 2011
Copyright© 2011 Claremont BioSolutions, LLC
1
Rapid Nucleic Acid extraction
• OmniLyse® Kit- miniature disposable
flow-through cell disruptor
• PureLyse® Kit- miniature disposable
flow-through cell disruptor plus
nucleic acid extraction
• RapidLyser™ - benchtop flow-through
instrument for mechanical cell lysis
and nucleic acid extraction
Copyright© 2011 Claremont BioSolutions, LLC
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Advantages of PureLyse®
Sample Prep
Typical NA extractions from
whole cells:
• 10 to 18 steps
• 1 to 2 hours
PureLyse® NA extraction:
•Lysis and extraction < 5 min
•Simple 2-step protocol
•Miniaturized
Copyright© 2011 Claremont BioSolutions, LLC
3
PureLyse® Prep vs. Qiagen DNeasy
PureLyse vs Qiagen; E. coli, 1ml samples
40
PureLyse
35
Qiagen
CT values
30
PL NTC
25
Q NTC
20
15
10
5
0
1.E+00
CFUs in
Sample
200,000,000
20,000,000
2,000,000
200,000
20,000
2,000
Copies in
PCR
5,000,000
500,000
50,000
5,000
500
50
0
1.E+02
1.E+04
1.E+06
1.E+08
CFUs/ ml
Sample: 1 ml of E.coli ,varying concentrations (n=3)
Lysis 1 min, Elution 1 min. 200 µl, 2x, 5ul to PCR
Copyright© 2011 Claremont BioSolutions, LLC
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PureLyse® Prep 1x108 CFU E.coli:
PCR analysis
PureLyse® Prep - 1 mL
sample
1x108 CFUs E.coli
2 minute lysis
1 minute elution, 200 µl
Real-time PCR results
>70% extraction efficiency
Sample
gDNA 10e7
PL 200
PL150
gDNA 10e4
PL 200 neg
PL150 neg
TE 8.0 neg
TE 8.8 neg
replicates
2
10
10
2
2
2
4
2
CV
0.1
1.4
2.6
0.0
1.1
1.2
1.3
1.0
interpolated copies
150
200
8,925,897 9,003,203
Copyright© 2011 Claremont BioSolutions, LLC
efficiency
150
200
71%
72%
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PureLyse® Prep Low Limit of
Detection
PureLyse® Prep to PCR E. coli titration
45
40
6.25 12.5
35
25
1 ml E.coli, 2.5 minute lysis
200 µl elution, 1 minute
125
1250
30
Ct
25
20
PureLyse® Prep
15
Achievable detection:
50 CFUs (6.25 CFU/PCR
reaction)
gDNA
10
5
0
1
10
100
1000
copies transfered to PCR
10000
CFUs in
PureLyse® prep
50
100
200
1000
10000
Copyright© 2011 Claremont BioSolutions, LLC
transferred to
PCR
6.25
12.5
25
125
1250
Efficiency
83%
67%
49%
33%
55%
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Detection of Bacillus subtilis in Synthetic Stool Mix:
PureLyse® sample prep to PCR
PureLyse®
in
PureLyse® to
to PCR; Bacillus
Bacillus subtilis
PureLyse®
subtilis in
in
Synthetic
Mix
Synthetic Stool Mix
60
125 CFUs, 50%
50
1250 CFUs, 100%
Ct
40
20
PureLyse®
PureLyse®
PureLyse®
PureLyse®
PureLyse®
NTC
PureLyse®
NTC
PureLyse® NTC
PureLyse®
EluElu
11
PureLyse®
PureLyse®
PureLyse®
1
Qiagen
EluElu
1 Elu
Qiagen
1NTC
10
PureLyse®
day
44
PureLyse®
day
Qiagen Elu 1
Qiagen
day
44
Qiagen
day
30
Swab SSM
0
1.E+00
1.E+00
1.E+01
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
CFUs
Copyright© 2011 Claremont BioSolutions, LLC
Copies in
PCR
0
12.5
1.25E+02
1.25E+03
1.25E+04
1.25E+05
1.25E+06
CFUs in
PureLyse®
Prep
0
100
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
n
6
6
6
5
5
5
5
Sample: 1 ml of
B. subtilis,varying
concentrations
(n=3) Lysis 2 min,
Elution 1 min.
200 µl, 2x, 25ul to
PCR
PureLyse®Prep: Mycobacterium bovis BCG
PureLyse® Prep vs Biospec/Qiagen to PCR
Mycobacterium bovis BCG (n=3)
PureLyse® samples:
50
•500 µl lysis and DNA
capture – 3 minutes
•250 µl elution
•625 µl lysis Biospec
bead beater - 3 minutes
•500 µl processed via
Qiagen DNeasy Kit
•250 µl elution
PureLyse® Elution
BioSpec/Qiagen
Standards
40
Ct
Biospec samples:
PureLyse® Elution Neg
30
Biospec/Qiagen Neg
20
biospec
10
1
12.5 µl into PCR
Purelyse®
Prep
100
10000
1000000
M. bovis BCG Concentration (cells/ml)std curve
Data provided by Peter Vandeventer and Angelika Niemz, Keck Graduate Institute
Copyright© 2011 Claremont BioSolutions, LLC
CFUs/ml
100
200
1000
100000
10000000
200
1000
100000
10000000
100
1000
10000
100000
1000000
10000000
copies in PCR
2.5
5
25
2500
250000
6.25
31.25
3125
312500
1.25
12.5
125
1250
12500
125000
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Synthetic Stool Mix
Detection of Bacillus subtilis in
Synthetic Stool Mix
Dextran Sulfate
Bile Salts
Mucin, Bovine
Human Serum Albumin
Human gDNA
E. coli
60
PureLyse®
50
PureLyse® NTC
Qiagen 3
40
PureLyse 3
Ct
100 µl Synthetic stool
samples brought up to 1
ml in Binding Buffer
• Lysis - 2 minutes
• Elution -1 minute, 200 µl
• 25 µl into PCR
PureLyse®Prep to PCR; B.subtilis in
Synthetic Stool Mix
30
20
10
0
1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07
CFUs
100% negative - true neg.
50 % of the samples at 100 CFUs (12.5/PCR) amplified as true
positives
Comparable to Qiagen Stool Kit
Copies in
PCR
0
12.5
1.25E+02
1.25E+03
1.25E+04
1.25E+05
1.25E+06
CFUs in
PureLyse®
Prep
0
100
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
Copyright© 2011 Claremont BioSolutions, LLC
Data provided by Boris Gites, Dipika Tuteja Shringarpure, Ivan Ban and Farhan Bukhari, Keck Graduate Institute
n
6
6
6
5
5
5
5
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PureLyse® Efficiency
% of DNA MEASURED
PureLyse® DNA extraction
of 1ml Sample (E. coli 1 x 108 cells)
100
90
80
70
60
50
40
30
20
10
0
89 %
79 %
54 %
7%
Flow-thru
n=5
93 %
Elution 1
Elution 1+2
Elution
1+2+3
Sample fractions
(following RNase A treatment)
Elution
1+2+3+4
Figure 1. Pico Green determination of DNA
yield (% predicted from cell count)
• flow-through and four 150 µl elutions
• 1 µg RNAse A treatment
• Average yield of all fractions: 88%
• 82% of DNA in the sum of four elutions
Figure 2. Pico Green measurement of NA in
the flow through and the eluant before and
after RNAse A digestion
• Reference DNA (gold), predicted by cell
count
• NA before RNAse: 82%
• NA after RNAse: 93%
Copyright© 2011 Claremont BioSolutions, LLC
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OmniLyse ® Prep:
Lysis of bacterial cells and spores
B. subtilis spores
E. coli cells
OmniLyse® Lysis---Qiagen preps--- PCR
250
Efficiency
200
150
100
FT
Elu1
50
Elu2
0
standard
Omnilyse no
R'nase
Omnilyse + R'nase standard + R'nase
Qiagen
OmniLyse
Treatment
OmniLyse® Lysis Enhances Performance of
Qiagen Kit, E. coli
OmniLyse® Prep: Lysis of Bacillus subtilis spores
• 500 µl samples (12.5 µl into PCR)
• Sample concentrations: 1,000, 100,000 and
100,000,000 spores
de Boer RR. Improved detection of microbial DNA after bead-beating before DNA isolation. J Microbiol
Methods 2010;80(2):209-11.
Copyright© 2011 Claremont BioSolutions, LLC
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Rapid Protein Purification
Copyright© 2011 Claremont BioSolutions, LLC
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OmniLyse® Sample Preparation:
50 mL Culture
Benefits of using OmniLyse® Prep
 Time savings - lysis to purification in <12 minutes
 Eliminates the need for lysing chemicals, lysozyme
or DNase treatment
 Disposable
 Ideal for the lab bench, the hood, the cold room or
in the field
 Easily adapted to variety of chromatography
products
 Can process a bacterial pellet from 50 mL culture
 Lyses Yeast in 2 minutes
Copyright© 2011 Claremont BioSolutions, LLC
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HisExpress™ Column:
Rapid and Simple Purification of His-tagged Proteins
< 5 min
< 5 min
Copyright© 2011 Claremont BioSolutions, LLC
Native or Denaturing conditions
Pipette friendly
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OmniLyse® Prep to HisExpress™ Affinity
tag Purification of GFP - His Tag
250
150
100
75
50
37
25
15
M L
FT
W1 W2 W3 E1
E2
A pellet from 50 mL of E. coli
expressing 6xHis-EmGFP was
prepared using OmniLyse® prep
for 3 min
6xHis-EmGFP (Invitrogen) was
purified under native conditions
using HisExpress™ Column in <5
min.
E3
Benefits of using OmniLyse™
 Time savings - lysis to purification
in <15 minutes
 Eliminates the need for lysozyme
or DNase treatment
 Disposable
 Ideal for the lab bench, the hood,
the cold room or in the field
 Easily adapted to variety of
chromatography products
Copyright© 2011 Claremont BioSolutions, LLC
Fluorescence units
20
80000
60000
40000
20000
0
Figure 3. GFP fluorescence remains stabile
during lysis. 6xHis-EmGFP fluorescence was
measured at 487 nm (ex) and 507 nm (em).
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Lysed samples from OmniLyse™, OmniLyse™
low binding (LB) and sonication were
HisExpress™ Column out performs competitor
in Yield, Purity, Time and Price
Purification of His-GFP using HisExpress™
Column or Qiagen Ni-NTA spin columns
250
150
100
75
 OmniLyse® Lysis preceded both methods
50
 OmniLyse® kit samples processed 3 minutes
37
His-GFP
25
 Qiagen samples purified as per manufacturer’s
instruction
Company
Total
Yield
(mg)
Purity
%
Sample
Prep
Time
Purification
Time
HisExpress™
Column
1.2
>95
<5 min
<5 min
Qiagen
spin column
0.4
>95
Variable
1 hr
Copyright© 2011 Claremont BioSolutions, LLC
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15
10
M HE Q
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His-PureLyse™ Prep: Rapid lysis and purification of
His-tagged protein in a single device
His-PureLyse™ sample preparation expedites lysing and purifying
Hi-tagged proteins from a sample in less than 5 minutes.
•Bacteria expressing 6xHis-EmGFP were lysed for 3 minutes in the
His-PureLyse™ Cartridge, followed by wash and elution steps.
•Fractions analyzed by SDS-PAGE and Coomassie staining:
M=marker, L=lysate, W=wash and E=elution.
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Configurations
HisExpress™
Column
HisPurLyse™
Prep
HisExpress™ micro DNA express™
column
Copyright© 2011 Claremont BioSolutions, LLC
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Acknowledgements
CBS
Bob Doebler
Tanya Ferguson
Margaret Morgan
Mark Brown
Gary Blackburn
Jim Sterling
Ali Nadim
KGI
Angelika Niemz
Peter Vanderventer
Barbara Erwin
Boris Gites
Dipika Tuteja Shringarpure
Ivan Ban
Farhan Bukhari
Copyright© 2011 Claremont BioSolutions, LLC
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