For Genetic Reseach
1.
2.
3.
4.
5.
Lambda Exonuclease
10×Lambda Exonuclease Buffer
4×Hybridization Buffer
Duplex-specific nuclease
2×Duplex-specific nuclease Buffer
6. ExonucleaseⅠ
7. 10×ExonucleaseⅠ Buffer
8. Ethachinmate
9. Stop Solution
10. 3mol/L Sodium Acetate, pH 5.2
DsDD cDNA Subtraction Kit Wako
Kit Contents (for 5 reactions)
Product Appearance
5μL×1 tube
25μL×1 tube
25μL×1 tube
5μL×1 tube
25μL×1 tube
5μL×1
25μL×1
50μL×1
50μL×1
200μL×1
tube
tube
tube
tube
tube
DsDD cDNA Subtraction Kit Wako
Wako Catalog No. 294-62001
Simple and quick enrichment of specifically expressed genes
Storage Condition ：−20℃
Product
Product Name
Catalog No.
Package Size
294-62001
for 5 reactions
Catalog No.
Package Size
Lambda Exonuclease
290-61501
1,000units
Exonuclease Ⅰ
294-61401
4,000units
dNTP Mixture
043-29291
0.2mL
311-90151
250mL
052-07221
100mL
054-07225
500mL
[Wako Chemicals USA]
546-01651
30μg
Agarose S
[NIPPON GENE CO., LTD.]
312-01193
100μg
50×TAE
[NIPPON GENE CO., LTD.]
313-90035
500mL
6×Loading Buffer Triple Dye
[NIPPON GENE CO., LTD.]
314-90261
1mL×3
Ethidium Bromide Solution
[NIPPON GENE CO., LTD.]
315-90051
10mL
Distilled Water, Deionized, Sterile
[NIPPON GENE CO., LTD.]
316-90101
100mL
DsDD cDNA Subtraction Kit Wako
Related Products
Product Name
Phenol/ Chloroform/ Isoamyl Alcohol (25 : 24 : 1)
[NIPPON GENE CO., LTD.]
Ethanol (99.5)
100bp DNA Step Ladder (100-1.5kbp)
Only 2 Day Performance
Listed products are intended for laboratory research use only.
Please visit our online catalog to search for other products from Wako; http://search.wako-chem.com/
■ A license agreement is required for purchase and use of this kit for commercial purposes.
■ The DsDD cDNA Subtraction Kit Wako is patent pending.
■
■
Wako Pure Chemical Industries, Ltd.
–– http://www.wako-chem.co.jp ––
1-2, Doshomachi 3-Chome,
Chuo-Ku, Osaka 540-8605, Japan
Tel: +81-6-6203-3741
Fax: +81-6-6201-5964
Online Cat.: http://search.wako-chem.com
Wako Chemicals USA, Inc.
–– http://www.wakousa.com ––
Wako Chemicals GmbH
–– http://www.wako-chemicals.de ––
Head Office (Richmond, VA):
Toll-Free (U.S. only): 1-877-714-1920
Tel: 1-804-714-1920/Fax: 1-804-271-7791
European Office:
Nissanstraße 2, D-41468
Neuss, Germany
Los Angeles Sales Office (Irvine, CA):
Tel: 1-949-679-1700/Fax: 1-949-679-1701
Tel: 49-2131-311-0
Fax: 49-2131-311100
Boston Sales Office (Cambridge, MA):
Tel: 1-617-354-6772/Fax: 1-617-354-6774
Start with an established cDNA Library
05803IBK
Recovery of Intact Tester cDNA
World’s First New Subtraction Kit Using Enzyme Digestion
Subtraction Efficiency [2]
The amplification levels of the highly expressed housekeeping genes
(GAPDH) and genes expressed differentially in hepatoma cells (AFP) were
compared by real-time PCR. Subtraction efficiency was determined by the
∆Ct value obtained by subtracting the Ct value of HepG2 cDNA from the
subtracted cDNA.
ΔCt value
(difference in cycle number)
12
From Physical Absorption Method to DsDD
The subtraction hybridization method is a powerful technique used to identify genes that are specifically expressed in tissue,
cell type or at a specific stage. Several methods, including physical absorption, have been used. Traditional procedures often
had several drawbacks such as being complex and labor-intensive, time-consuming, and technically demanding. They required
several rounds of hybridization and took about 4 days. They also required the Tester and Driver cDNA to be prepared each time
from mRNA because the cDNA Library could not be used as the starting material due to the influence of vector-derived sequences.
The DsDD cDNA Subtraction Kit Wako (patent pending) is a method based on
"Duplex-specific Direct Digestion" (DsDD), which overcomes traditional
subtraction methods. The Tester and Driver cDNA form drawbacks of hybrids of
genes, that are expressed nonspecifically. After hybridization, the hybrid ds
cDNA from Tester and Driver cDNA are digested by duplex-specific
nuclease. Finally, the Driver cDNA is removed by Exonuclease. This method
efficiently enriches cDNA specifically expressed in Tester cDNA.
For example, when analyzing specific functions and properties of cancer cell
tissue, the Tester is prepared from cancer tissue and the Driver from normal
tissue, then cDNA derived from cancer specified gene is enriched. The DsDD
cDNA Subtraction Kit Wako is a revolutionary technique that uses the cDNA
Library as starting material, and the subtracted Tester cDNA can be recovered
intact.
10
10.315
8
6
4
The subtracted cDNA of the highly expressed housekeeping GAPDH
genes dswere detected 10 cycles later than the HepG2 cDNA. The AFP
genes, which are expressed specifically in HepG2, were detected after a
similar number of cycles. These results show that GAPDH is less than
1/1,000 (1/210.315) and that highly efficient subtracted cDNA were
constructed.
2
0
−0.26
GAPDH
AFP
−2
Real-Time PCR Reaction Conditions
What is Subtraction?
Example）
cDNA（1ng/μL）
5μL
2×Mastermix
50℃ 2min.
12.5μL
Sense Primer（5μM）
↓
1.5μL
95℃ 10min.
Tester
Driver
Hepatoma cells / tissues
Normal liver cells / tissues
Antisense Primer（5μM）
A. Housekeeping genes
A. Housekeeping genes
SYBR® Green I
0.75μL
95℃ 15sec.
dH2O
3.75μL
60℃ 1min.
B. Liver-specific genes
−
1.5μL
Total 25μL
B. Liver-specific genes
(A,B,C)−（A,B) = C
↓ 40cycles
Subtracted cDNA [B]
[B]‒[A]
GAPDH
13.651
23.966
10.315
AFP
17.884
17.624
−0.26
Principle
Tester Library (abnormal)
Driver Library (normal)
A Tester cDNA Library and a Driver cDNA Library are
prepared. In the cDNA Library for the Tester, the cDNA
should be inserted in the same direction as the vector.
Target
gene
Features
＊1
DNA amplification
(such as PCR)
DNA amplification
(such as PCR)
Amplified Tester dsDNA from Library
Duplex-specific nuclease is purified from Kamchatka crab hepatopancreas
5‘
3‘
5‘
3‘
3‘
-P-5‘
AAAAA
TTTTT
3‘
-P-5‘
5‘
3‘
5‘
3‘
AAAAA
TTTTT
AAAAA
TTTTT
＊1
3‘
5‘
3‘
5‘
Proper Restriction
Endonuclease Treatment
5‘
3‘
AAAAA
TTTTT
3‘
-P-5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
5‘
3‘
AAAAA
TTTTT
3‘
-P-5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
＊3
＊4
1:200
Driver
Tester-Driver
Normal liver tissue cDNA
Subtracted cDNA
AFP
GAPDH
AFP
GAPDH
5‘
AFP
AAAAA
TTTTT
3‘
5‘
5‘
AAAAA
5‘
3‘
TTTTT
AAAAA
3‘
5‘
AAAAA
3‘
3‘
5‘
AAAAA
3‘
3‘
TTTTT
5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
3‘
5‘
←1,500bp
←1,000bp
AFP : alpha-fetoprotein
GAPDH : glyceraldehyde-3-phosphate dehydrogenase
1% Agarose S
The existence of highly expressed housekeeping GAPDH genes are confirmed by PCR amplification of HepG2 cDNA and
normal liver tissue cDNA, whereas not in subtracted cDNA.
Meanwhile, AFP genes, specifically expressed in HepG2 cells, were not confirmed with normal liver tissue cDNA, but in the
similar amplification level, the existence was confirmed with subtracted cDNA.
TTTTT
＊5
5‘
3‘
AAAAA
TTTTT
5‘
3‘
AAAAA
TTTTT
5‘
3‘
AAAAA
TTTTT
5‘
3‘
TTTTT
3‘
5‘
5‘
AAAAA
3‘
5‘
AAAAA
3‘
3‘
5‘
3‘
TTTTT
5‘
3‘
5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
Subtracted cDNA
Duplex-specific nuclease Treatment
Enzymes that specifically cleave ds DNA are used. A
high reaction temperature (68℃) makes reactions highly
specific. After the reaction, only the single-stranded
DNA containing the Tester’s specifically expressed
genes will remain.
After DNA amplification,
ExonucleaseⅠ Treatment＊6
Target gene
931 bp→
859 bp→
5‘
3‘
Hybridization
The Tester cDNA and Driver cDNA react together at
68℃ for 16 to 20 hours (mixing ratio = 1:200) Most of
the Tester cDNA form ds DNA due to the excessive
amounts of Driver cDNA.
After Hybridization , Duplex-specific
nuclease Treatment＊5
Target gene
Tester
Restriction Endonuclease Treatment
The digestion of adapters on both ends ensures
accurate hybridization results.
＊4
HepG2 cDNA
Lambda Exonuclease Treatment
The double-stranded DNA’s 5’ phosphorylated primer is
recognized and is degraded by a 5’ to a 3’. Subtraction
efficiency is enhanced as there is no hybridization of
Testers.
＊3
Lambda Exonuclease
Treatment
cDNA Library from human hepatoma cell line HepG2 and human normal liver tissue are used as templates. The subtraction was
performed using the DsDD cDNA Subtraction Kit Wako. The evaluation of the subtracted cDNA was conducted using highly
expressed housekeeping GAPDH genes and AFP genes, specifically expressed in HepG2 cells, as control.
PCR amplification of HepG2 cDNA (Tester ss cDNA), normal liver tissue cDNA (Driver ds cDNA), and subtracted cDNA was
performed, each sample being 1ng, and the results were analyzed by electrophoresis.
＊2
Amplified Driver dsDNA from Library
AAAAA
TTTTT
＊2
Subtraction Efficiency [1]
GAPDH
Tester cDNA Amplification
During DNA amplification, a primer with phosphoric acid
added to the 5’ end is used.
Target gene
■ Start with established cDNA Libraries
■ Intact recovery of the subtracted Tester cDNA
■ Adoption of duplex-specific nuclease digestion
■ 2 day performance
ΔCt value
HepG2 cDNA [A]
1℃ per minute from 60℃ to 95℃
C. Hepatoma-specific
genes
Subtraction is an efficient gene analysis method that
enriches the amount of differentially expressed genes
by subtracting the expressed genes present in both
the Driver cDNA and the Tester cDNA.
Cycle Number
↓
＊6
ExonucleaseⅠ Treatment
Enzymes that specifically cleave single-stranded DNA
are used. Non-specific genes are single stranded, thus
degraded.
This highly efficient cDNA subtraction method
gives results in only 2 days.
World’s First New Subtraction Kit Using Enzyme Digestion
Subtraction Efficiency [2]
The amplification levels of the highly expressed housekeeping genes
(GAPDH) and genes expressed differentially in hepatoma cells (AFP) were
compared by real-time PCR. Subtraction efficiency was determined by the
∆Ct value obtained by subtracting the Ct value of HepG2 cDNA from the
subtracted cDNA.
ΔCt value
(difference in cycle number)
12
From Physical Absorption Method to DsDD
The subtraction hybridization method is a powerful technique used to identify genes that are specifically expressed in tissue,
cell type or at a specific stage. Several methods, including physical absorption, have been used. Traditional procedures often
had several drawbacks such as being complex and labor-intensive, time-consuming, and technically demanding. They required
several rounds of hybridization and took about 4 days. They also required the Tester and Driver cDNA to be prepared each time
from mRNA because the cDNA Library could not be used as the starting material due to the influence of vector-derived sequences.
The DsDD cDNA Subtraction Kit Wako (patent pending) is a method based on
"Duplex-specific Direct Digestion" (DsDD), which overcomes traditional
subtraction methods. The Tester and Driver cDNA form drawbacks of hybrids of
genes, that are expressed nonspecifically. After hybridization, the hybrid ds
cDNA from Tester and Driver cDNA are digested by duplex-specific
nuclease. Finally, the Driver cDNA is removed by Exonuclease. This method
efficiently enriches cDNA specifically expressed in Tester cDNA.
For example, when analyzing specific functions and properties of cancer cell
tissue, the Tester is prepared from cancer tissue and the Driver from normal
tissue, then cDNA derived from cancer specified gene is enriched. The DsDD
cDNA Subtraction Kit Wako is a revolutionary technique that uses the cDNA
Library as starting material, and the subtracted Tester cDNA can be recovered
intact.
10
10.315
8
6
4
The subtracted cDNA of the highly expressed housekeeping GAPDH
genes dswere detected 10 cycles later than the HepG2 cDNA. The AFP
genes, which are expressed specifically in HepG2, were detected after a
similar number of cycles. These results show that GAPDH is less than
1/1,000 (1/210.315) and that highly efficient subtracted cDNA were
constructed.
2
0
−0.26
GAPDH
AFP
−2
Real-Time PCR Reaction Conditions
What is Subtraction?
Example）
cDNA（1ng/μL）
5μL
2×Mastermix
50℃ 2min.
12.5μL
Sense Primer（5μM）
↓
1.5μL
95℃ 10min.
Tester
Driver
Hepatoma cells / tissues
Normal liver cells / tissues
Antisense Primer（5μM）
A. Housekeeping genes
A. Housekeeping genes
SYBR® Green I
0.75μL
95℃ 15sec.
dH2O
3.75μL
60℃ 1min.
B. Liver-specific genes
−
1.5μL
Total 25μL
B. Liver-specific genes
(A,B,C)−（A,B) = C
↓ 40cycles
Subtracted cDNA [B]
[B]‒[A]
GAPDH
13.651
23.966
10.315
AFP
17.884
17.624
−0.26
Principle
Tester Library (abnormal)
Driver Library (normal)
A Tester cDNA Library and a Driver cDNA Library are
prepared. In the cDNA Library for the Tester, the cDNA
should be inserted in the same direction as the vector.
Target
gene
Features
＊1
DNA amplification
(such as PCR)
DNA amplification
(such as PCR)
Amplified Tester dsDNA from Library
Duplex-specific nuclease is purified from Kamchatka crab hepatopancreas
5‘
3‘
5‘
3‘
3‘
-P-5‘
AAAAA
TTTTT
3‘
-P-5‘
5‘
3‘
5‘
3‘
AAAAA
TTTTT
AAAAA
TTTTT
＊1
3‘
5‘
3‘
5‘
Proper Restriction
Endonuclease Treatment
5‘
3‘
AAAAA
TTTTT
3‘
-P-5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
5‘
3‘
AAAAA
TTTTT
3‘
-P-5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
＊3
＊4
1:200
Driver
Tester-Driver
Normal liver tissue cDNA
Subtracted cDNA
AFP
GAPDH
AFP
GAPDH
5‘
AFP
AAAAA
TTTTT
3‘
5‘
5‘
AAAAA
5‘
3‘
TTTTT
AAAAA
3‘
5‘
AAAAA
3‘
3‘
5‘
AAAAA
3‘
3‘
TTTTT
5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
3‘
5‘
←1,500bp
←1,000bp
AFP : alpha-fetoprotein
GAPDH : glyceraldehyde-3-phosphate dehydrogenase
1% Agarose S
The existence of highly expressed housekeeping GAPDH genes are confirmed by PCR amplification of HepG2 cDNA and
normal liver tissue cDNA, whereas not in subtracted cDNA.
Meanwhile, AFP genes, specifically expressed in HepG2 cells, were not confirmed with normal liver tissue cDNA, but in the
similar amplification level, the existence was confirmed with subtracted cDNA.
TTTTT
＊5
5‘
3‘
AAAAA
TTTTT
5‘
3‘
AAAAA
TTTTT
5‘
3‘
AAAAA
TTTTT
5‘
3‘
TTTTT
3‘
5‘
5‘
AAAAA
3‘
5‘
AAAAA
3‘
3‘
5‘
3‘
TTTTT
5‘
3‘
5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
5‘
3‘
AAAAA
TTTTT
3‘
5‘
Subtracted cDNA
Duplex-specific nuclease Treatment
Enzymes that specifically cleave ds DNA are used. A
high reaction temperature (68℃) makes reactions highly
specific. After the reaction, only the single-stranded
DNA containing the Tester’s specifically expressed
genes will remain.
After DNA amplification,
ExonucleaseⅠ Treatment＊6
Target gene
931 bp→
859 bp→
5‘
3‘
Hybridization
The Tester cDNA and Driver cDNA react together at
68℃ for 16 to 20 hours (mixing ratio = 1:200) Most of
the Tester cDNA form ds DNA due to the excessive
amounts of Driver cDNA.
After Hybridization , Duplex-specific
nuclease Treatment＊5
Target gene
Tester
Restriction Endonuclease Treatment
The digestion of adapters on both ends ensures
accurate hybridization results.
＊4
HepG2 cDNA
Lambda Exonuclease Treatment
The double-stranded DNA’s 5’ phosphorylated primer is
recognized and is degraded by a 5’ to a 3’. Subtraction
efficiency is enhanced as there is no hybridization of
Testers.
＊3
Lambda Exonuclease
Treatment
cDNA Library from human hepatoma cell line HepG2 and human normal liver tissue are used as templates. The subtraction was
performed using the DsDD cDNA Subtraction Kit Wako. The evaluation of the subtracted cDNA was conducted using highly
expressed housekeeping GAPDH genes and AFP genes, specifically expressed in HepG2 cells, as control.
PCR amplification of HepG2 cDNA (Tester ss cDNA), normal liver tissue cDNA (Driver ds cDNA), and subtracted cDNA was
performed, each sample being 1ng, and the results were analyzed by electrophoresis.
＊2
Amplified Driver dsDNA from Library
AAAAA
TTTTT
＊2
Subtraction Efficiency [1]
GAPDH
Tester cDNA Amplification
During DNA amplification, a primer with phosphoric acid
added to the 5’ end is used.
Target gene
■ Start with established cDNA Libraries
■ Intact recovery of the subtracted Tester cDNA
■ Adoption of duplex-specific nuclease digestion
■ 2 day performance
ΔCt value
HepG2 cDNA [A]
1℃ per minute from 60℃ to 95℃
C. Hepatoma-specific
genes
Subtraction is an efficient gene analysis method that
enriches the amount of differentially expressed genes
by subtracting the expressed genes present in both
the Driver cDNA and the Tester cDNA.
Cycle Number
↓
＊6
ExonucleaseⅠ Treatment
Enzymes that specifically cleave single-stranded DNA
are used. Non-specific genes are single stranded, thus
degraded.
This highly efficient cDNA subtraction method
gives results in only 2 days.
For Genetic Reseach
1.
2.
3.
4.
5.
Lambda Exonuclease
10×Lambda Exonuclease Buffer
4×Hybridization Buffer
Duplex-specific nuclease
2×Duplex-specific nuclease Buffer
6. ExonucleaseⅠ
7. 10×ExonucleaseⅠ Buffer
8. Ethachinmate
9. Stop Solution
10. 3mol/L Sodium Acetate, pH 5.2
DsDD cDNA Subtraction Kit Wako
Kit Contents (for 5 reactions)
Product Appearance
5μL×1 tube
25μL×1 tube
25μL×1 tube
5μL×1 tube
25μL×1 tube
5μL×1
25μL×1
50μL×1
50μL×1
200μL×1
tube
tube
tube
tube
tube
DsDD cDNA Subtraction Kit Wako
Wako Catalog No. 294-62001
Simple and quick enrichment of specifically expressed genes
Storage Condition ：−20℃
Product
Product Name
Catalog No.
Package Size
294-62001
for 5 reactions
Catalog No.
Package Size
Lambda Exonuclease
290-61501
1,000units
Exonuclease Ⅰ
294-61401
4,000units
dNTP Mixture
043-29291
0.2mL
311-90151
250mL
052-07221
100mL
054-07225
500mL
[Wako Chemicals USA]
546-01651
30μg
Agarose S
[NIPPON GENE CO., LTD.]
312-01193
100μg
50×TAE
[NIPPON GENE CO., LTD.]
313-90035
500mL
6×Loading Buffer Triple Dye
[NIPPON GENE CO., LTD.]
314-90261
1mL×3
Ethidium Bromide Solution
[NIPPON GENE CO., LTD.]
315-90051
10mL
Distilled Water, Deionized, Sterile
[NIPPON GENE CO., LTD.]
316-90101
100mL
DsDD cDNA Subtraction Kit Wako
Related Products
Product Name
Phenol/ Chloroform/ Isoamyl Alcohol (25 : 24 : 1)
[NIPPON GENE CO., LTD.]
Ethanol (99.5)
100bp DNA Step Ladder (100-1.5kbp)
Only 2 Day Performance
Listed products are intended for laboratory research use only.
Please visit our online catalog to search for other products from Wako; http://search.wako-chem.com/
■ A license agreement is required for purchase and use of this kit for commercial purposes.
■ The DsDD cDNA Subtraction Kit Wako is patent pending.
■
■
Wako Pure Chemical Industries, Ltd.
–– http://www.wako-chem.co.jp ––
1-2, Doshomachi 3-Chome,
Chuo-Ku, Osaka 540-8605, Japan
Tel: +81-6-6203-3741
Fax: +81-6-6201-5964
Online Cat.: http://search.wako-chem.com
Wako Chemicals USA, Inc.
–– http://www.wakousa.com ––
Wako Chemicals GmbH
–– http://www.wako-chemicals.de ––
Head Office (Richmond, VA):
Toll-Free (U.S. only): 1-877-714-1920
Tel: 1-804-714-1920/Fax: 1-804-271-7791
European Office:
Nissanstraße 2, D-41468
Neuss, Germany
Los Angeles Sales Office (Irvine, CA):
Tel: 1-949-679-1700/Fax: 1-949-679-1701
Tel: 49-2131-311-0
Fax: 49-2131-311100
Boston Sales Office (Cambridge, MA):
Tel: 1-617-354-6772/Fax: 1-617-354-6774
Start with an established cDNA Library
05803IBK
Recovery of Intact Tester cDNA