Nucleic Acid Purification
System
General Information
−
Purification column
●
Specially treated glass filter membrane
●
Binds DNA efficiently under high salt
●
Mini--column spin technology
Mini
●
●
Optimized buffer to ensure only Nucleic Acid is
isolated
Water or low salt buffers (appropriate pH) to
elute highly pure nucleic acid.
FEATURES
●
High yield, reliable and reproducible
●
High purity
●
Fast and Easy purification
●
No toxic or organicorganic-based extraction
●
Wide applications − restriction digestion, cloning, PCR, DNA sequencing etc.
●
Column design – colored ring (BLUE
(BLUE
BLUE)) to aid visualization and better aiming
GENOMIC DNA EXTRACTION - GENERAL OVERVIEW
1) Sample Disruption (2 steps or single step)
•
Chemical Lysis
- Detergent
- Protein Denaturant
●
●
Guanidine Salt
Proteinase K
2) Addition of Ethanol – Nucleic Acid Binding Enhancer
3) Nucleic Acid Binding
4) Column Washing 1 – Inhibitor Removal (Optional)
5) Column Washing 2 – Ethanol Wash
6) Column Drying – important step
7) Elution
Comparison analysis for Tissue DNA Extraction Kit
M
V
P
Q
M
Chicken muscle tissue
V
P Q
M
Fish muscle tissue
V
49.3
1.89
1.45
P
17.5
1.77
2.24
Q
49.4
1.93
V
10.5
1.69
1.48
P
4.8
1.85
Q
18.6
V
13.8
1.80
P
7.0
Q
10.1
Chicken heart tissue
A260/280
A260/280
A260/280
M : VC Lamda/Hind III DNA Marker
V: Vivantis
P: Supplier P
Q: Supplier Q
M
P Q
ng/ul
ng/ul
ng/ul
V
V
Q
Cultured NS1 myeloma
ng/ul
A260/280
V
49.3
1.89
Q
47.5
1.77
Comparison analysis on down stream applications
PCR
M
V
P
Q
M : VC 100bp Plus DNA Ladder
V: Vivantis
P: Supplier P
Q: Supplier Q
PCR amplification of β-actin gene from
fish muscle tissue
Comparison with Qiagen
Features
GF-1 Tissue DNA
Extraction Kit
QIAGEN (DNeasy Blood and
Tissue Kit)
Incubation
temperature
65ºC with Proteinase K.
Optimum temperature for
Proteinase K is 60-70ºC
56ºC with Proteinase K
Sample size needed
for each purification
20mg
25mg
Maximum loading
volume
900µl. Avoid contamination
on cap
700µl
Elution volume
200µl. Total yield may not be
too high but the
concentration is suitable for
downstream applications
2 x 200µl. Total yield of DNA
may be higher but the
concentration of DNA may be
too low for downstream
applications
Comparison analysis for Blood DNA Extraction Kit
Human Genomic DNA extracted from
200ul of FRESH blood.
V
Human Genomic DNA extracted from
200ul of FROZEN blood.
Q
V
Legend:
V = VIVANTIS
Q = Supplier Q
Lane 1 = Control Marker
Q
Comparison with Qiagen
Features
GF-1
1 Blood DNA Extraction
Kit
QIAGEN (QIAamp DNA Blood
Mini Kit)
Enzyme used
Proteinase K
Protease: Protease degrade
easily in the present of EDTA at
concentration >8mM. EDTA is
the most common
anticoagulant for blood samples
Incubation temperature
65ºC. This is the optimum
temperature for Proteinase K
activity.
56ºC.
Washing step
3 washing steps to make sure
salts are removed completely
2 washing steps.
Elution volume
100µl. Total yield may not be
too high but the concentration
is suitable for downstream
applications
2 x 200µl. Total yield of DNA
may be higher but the
concentration of DNA may be
too low for downstream
applications
Plant DNA Extraction Kit
Q V
DNA extracted from
20mg of leaf tissue
RNA contamination
V: Vivantis
Q: Supplier Q
Comparison with Qiagen
Features
GF-1
1 Plant DNA Extraction
kit
QIAGEN (DNeasy Plant kit)
Starting material
Up to 30mg
Up to 100mg
Sample types
Leaf, flower, root, seed and stem
Leaf, flower, root, seed, stem,
Not applicable on fungi
fungi and spores
Liquid nitrogen is recommended
Liquid nitrogen, TissueRuptor,
Tissue disruption
Tissuelyser system is
recommended
Sample Lysis
Proteinase K aided
Non-enzymatic lysis. Additional
precipitation step to remove
protein, cell debris and
polysaccharides.
Cell debris removal
Removed by centrifugation
Removed by centrifugation and a
filtration column.
Maximum loading volume
900µl
700µl
Binding buffer
Binding buffer and ethanol are
Binding buffer is added with
added separately to the sample
ethanol prior loading to column
Bacteria DNA Extraction Kit – 1ml bacteria culture
V
V: Vivantis
Q
Q: Supplier Q
Comparison with Qiagen
Features
GF-1
1 Bacteria DNA Extraction
Kit
Lysoyme treatment Lysozyme treatment for both
Gram negative and Gram
positive bacteria
QIAGEN (DNeasy Blood and
Tissue Kit)
Lysozyme treatment for Gram
positive bacteria only.
Lysis
2-step
step lysis with Proteinase K for 2-step lysis for Gram negative
both Gram negative and Gram
bacteria and single step lysis
positive bacteria
for Gram positive bacteria
(both with Proteinase K)
Incubation
temperature
65ºC with Proteinase K.
Optimum temperature for
Proteinase K is 60-70ºC
56ºC with Proteinase K
Elution volume
100µl. Total yield may not be too
high but the concentration is
suitable for downstream
applications
200µl. Total yield of DNA may
be higher but the
concentration of DNA may be
too low for downstream
applications
Improvement and comparison analysis for
Plasmid DNA Extraction kit
Improvements:
●
Smaller working volume, less working steps (1 time spinning), shorter purification time
Current 1.4ml
NEW: 0.9ml
●
Better precipitation, better handling
M
V
P
M
Q
V
P
Q
M
V
P
Q
M : VC 1kb DNA Ladder
V: Vivantis
P: Supplier P
Q: Supplier Q
pTZ57R –high copy, 2986bp
ng/ul
pCAMBIA 1302 –high
high copy, 10549bp
ng/ul
A260/280
pBR322 –low copy, 4631bp
A260/280
ng/ul
A260/280
V
134.7
1.98
V
210.2
1.93
V
63.5
1.97
P
108.3
1.92
P
133.2
1.91
P
68.3
1.87
Q
83.3
1.88
Q
174.3
1.90
Q
58.2
1.94
Comparison analysis on downstream applications
Restriction enzyme digestion
M1
V
P
Q
V
P
Q
V
P
PCR
Q
M2 -ve
V
P
Q
M1 : VC 1kb DNA Ladder
M2: VC 100bp Plus DNA Ladder
V: Vivantis
P: Supplier P
Undigested
Q: Supplier Q
plasmid/HindIII plasmid/FauNDI
pCAMBIA
Purified pCAMBIA is able to be
digested at comparable level.
Purified pTZ57R is able
to be amplified to obtain
product size 250bp by
using M13 forward/
reverse primers.
Comparison with Qiagen
Features
GF-1 Plasmid DNA
Extraction Kit
QIAGEN (QIAprep Miniprep)
Plasmid type
Low copy and high copy
number plasmids. Not tested
for cosmid
Low copy and high copy
number plasmids, cosmid
Mixing step
Invertion 6-10 times.
Thorough invertion leads to
well formation of protein
precipitate
Invertion 4-6 times.
Washing step
1 washing step
2 washing steps.
Elution volume
100µl. With higher elution
volume, more plasmid can be
eluted.
50µl. DNA concentration is
higher but total yield is lower.
Improvement and comparison analysis for
Gel DNA Recovery kit
●
●
Solubilization of agarose
− Addition of Buffer GB is reduced from 5 volumes to 1 volume.
Colourless Binding buffer - no pH adjustment is required.
M1 Ori V P Q
M1 Ori V P Q
Ori
V
P
Q
M2
Ori V P Q M2
Ori V
P
Q
M
5kb
10kb
362bp
1.5kb
M1: VC 100bp Plus DNA Ladder
V: Vivantis
P: Supplier P
M2: VC 1kb DNA Ladder
Q: Supplier Q
Ori: Original sample
5kb PCR product purification
Concentration
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
Purity
2.500
2.000
n g /u l
2 6 0 /2 8 0
1.500
1.000
0.500
0.000
1
2
3
4
average
Vivantis
Average concentration: 32.0 ng/ul
Average purity: 1.848
Total recovery: 1600ng
Recovery yield: 56%
1
2
3
4
average
Qiagen
concentration: 36.6ng/ul
Purity: 1.912
Total recovery: 1830ng
Recovery yield: 64%
Conclusion: Consistent
nsistent in terms of concentration and purity
Comparison with Qiagen
Features
GF-1
1 Gel DNA Recovery
Kit
QIAGEN (QIAquick SpinKit)
Buffer volume used
1 gel volume. More samples 3 gel volumes
can be processed
Use of isopropanol
Only for fragments <400bp
and >4kb
Apply on all fragment size.
PCR Clean-up Kit
Initial total DNA 3750ng
Concentration
120.0
Purity
2.000
100.0
1.500
2 6 0 /2 8 0
n g /u l
80.0
1.000
60.0
40.0
0.500
20.0
0.000
0.0
1.000
2
3
4
5
6
average
Average concentration: 71.2 ng/ul
Average purity: 1.748
1.000
2
3
4
5
6
average
Total recovery: 3560ng
Recovery Yield: 95%
Conclusion: Consistent
nsistent in terms of concentration and purity
Food DNA Extraction Kit – 100mg of food sample
PCR amplification of chloroplast DNA from extracted DNA
E
V
E
V
E : supplier E
V: Vivantis
Oat
Soy flour
Comparison with EURx
Features
GF-1
EURx (GeneMATRIX FoodExtract DNA Purification Kit
Types of samples
Dried plant, flour, , corn flakes,
soymilk, soysauce, cheese,
biscuits, wheat grains, bread,
chocolate powder, etc.
Dried plant, flour, , corn flakes,
soymilk, soysauce, etc.
Starting material
100-200mg
100-300mg
Washing step
3 washing steps to make sure
salts are removed completely
2 washing steps.
96-well
well DNA Extraction Kit
4 type of kits:
Genomic, Blood, Plasmid and PCR CleanClean
up
High throughput
96 minipreps simultaneously
Use centrifugation or vacuum manifold
Multichannel pipettes are recommended
Genomic DNA Kit – 1ml culture of bacteria E. coli
V
Q
V
Q
ng/ul
35.0
160
A260/280
1.93
2.09
Smearing and RNA contamination were
observed from Qiagen kit
RNA contamination
V: Vivantis
Q: supplier Q
Genomic DNA Kit – 10mg of animal tissue
M
V1 Q1
V2 Q2
V3 Q3
M : VC Lambda/HindIII DNA Marker
V: Vivantis
Q: supplier Q
1: Mouse liver
2. Mouse ear
3. Mouse kidney
Severe RNA contamination was
observed from Qiagen kit
V
Q
Mouse liver
ng/ul A260/280
60.2
1.97
67.7
2.09
V
Q
Mouse ear
ng/ul A260/280
29.1
1.72
25.0
1.91
V
Q
Mouse Kidney
ng/ul A260/280
35.8
1.94
58.0
2.1
Blood DNA Kit – 200µl frozen human blood
M
G
V
Q
ng/ul
A260/280
G
11.7
0.85
V
18.4
1.84
Q
25.4
2.21
Smearing was observed from Qiagen kit
M : VC Lambda/HindIII DNA Marker
V: Vivantis
Q: Supplier Q
G: supplier G
Plasmid DNA Kit
M
V1 Q1
V2 Q2
Plasmid 1 - pTZ57R
Plasmid 2 - pGEM
ng/ul A260/280
V1 33.4
1.90
Q1 29.8
1.72
ng/ul
V2 23.9
Q2 13.8
M : VC 1kb DNA Ladder
V: Vivantis
Q: supplier Q
A260/280
1.67
1.79
PCR Clean-up Kit
M
A
B
A
B
No significant lost of DNA
1.5kb
8kb
M : VC 1kb DNA Ladder
A: Before B: After
Comparison with Qiagen
Features
GF-1
QIAGEN
Shorter sample lysis
time
1-2 hours
Up to overnight
Flexible usage of
equipment
Use centrifugation or vacuum Use ONLY particular types of
manifold
centrifuge or QIAvac
Ease of handling
Use of Adhesive film for 96-well block sealing
More stable reagent
used
Proteinase K: Proteinase K is Protease: Protease degrade
stable in the present of EDTA easily in the present of EDTA
and detergent
at concentration >8mM. EDTA
is a common anticoagulant for
blood
Flexible membrane
drying step
Users can choose to use
centrifugation, vacuum or
incubation at 65°C for
drying
Centrifuge for 15 min for
ethanol evaporation by using
heat generated during
centrifugation
Less elution volume
30µl
60µl
Use 12 pieces of sealing cap
strips for 96-well block sealing
TOTAL RNA EXTRACTION KIT
Lyse sample by homogenization or enzymatic lysis
Loading to
Homogenization
column
Spin 13,000g, 2min
Addition of ethanol
Loading to RNA
Binding column
Wash 1x followed by DNase I treatement
Wash 3x
Elution
Total RNA Extraction from various tissues
Bacteria
1
2
V
Leaf
1
2
Liver
1
2
Spleen
Kidney
1
1
2
Q
Total RNA extraction from 1ml of bacteria culture
V: Vivantis
Q: supplier Q
2
DNA contamination analysis
Testing for genomic DNA contamination by amplification of bacteria 16S rRNA
RT-PCR
PCR
PCR only
Comparison with Qiagen
Features
GF-1
1 Total RNA Extraction Kit
QIAGEN (RNeasy Mini Kit)
Sample type
Bacteria, animal tissues, cultured
animal cells, plant, and yeast.
Bacteria, animal tissues, cultured
animal cells, plant, yeast and
filamentous fungi.
DNaseI treatment
Incorporated in the protocol
Optional protocol (need to
purchase DNaseI separately)
Blood Total RNA Extraction Kit – 400µl of human fresh blood
V
Q
DNA contamination
V: Vivantis
Q: Qiagen
Comparison with Qiagen
Features
Blood Total RNA Extraction
Kit
QIAGEN (QIAamp RNA Blood
Mini Kit)
Sample types
Fresh or frozen whole blood.
Fresh whole blood only.
Applicable on tissues and
cultured animal cells.
Starting material
Up to 1 ml
Up to 1.5ml
Sample lysis
Single step lysis with
Proteinase K
3 step lysis without Proteinase
K (2 x erythrocytes lysis,
leucocytes lysis)
DNaseI treatment
Incorporated in the protocol
Optional protocol (need to
purchase DNaseI separately)
Cell debris removal
By centrifugation
By a filtration column.
Viral Nucleic Acids Extraction Kit
●
●
Designed for rapid and efficient purification of viral RNA/DNA from
samples:
serum; plasma; body fluid or virus-infected
infected cell culture supernatant
●
Carrier RNA
●
●
Provided in the kit.
Spike into Buffer VL (lysis buffer) prior
purification. (Add 15µ
15µl of carrier RNA into 200µ
200µl
of buffer VL per reaction.)
●
Prepare in small aliquots and store at -20oC.
●
Only can be freezefreeze- thawed ONCE!
Comparison analysis for Viral Nucleic Acid Extraction Kit
RT-PCR
PCR analysis on dengue virus gene
Vivantis Kit
Supplier R Kit
Comparison analysis for Viral Nucleic Acid Extraction Kit
PCR analysis on adenovirus gene
Vivantis Kit
Supplier R Kit
Summary of comparison analysis fo
for Viral Nucleic Acid Extraction Kit
• Vivantis recovered
more Viral RNA than
Roche High Pure NA kit
(t-test: p<0.05)
• Roche High Pure
recovered more Viral
DNA but statistically
insignicant (t-test:
p>0.05)
Evaluation analysis performed by our local renowned university
New Products Launching
1. Total DNA extraction from petroleum based sample
2. Ambiclean Kit (PCR & Gel Clean up)
3. 96-well Tissue DNA kit
AmbiClean Kit (PCR & Gel)
M
A
P
A
P
M
Legend:
Legend:
M: VC 1kb DNA ladder
M: VC 1kb DNA ladder
A: PCR product before purification
A: PCR product before purification
P: Purified PCR product using Dual Gel &
PCR clean up kit (PCR clean up)
P: Purified PCR product using Dual Gel & PCR
clean up kit (Gel Recovery)
QUALITY CONTROL – FILTER SHEET
Lot Differentiation:
- 15 sheets/lot.
- 1 positive control/ lot
THANK YOU