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For Genetic Reseach

DsDD cDNA Subtraction Kit Wako


DsDD cDNA Subtraction Kit Wako
Kit Contents (for 5 reactions) Product Appearance
1. Lambda Exonuclease 5L1 tube
2. 10Lambda Exonuclease Buffer 25L1 tube
3. 4Hybridization Buffer 25L1 tube
Wako Catalog No. 294-62001
4. Duplex-specific nuclease 5L1 tube
5. 2Duplex-specific nuclease Buffer 25L1 tube
6. Exonuclease 5L1 tube
Simple and quick enrichment of specically expressed genes
7. 10Exonuclease Buffer 25L1 tube
8. Ethachinmate 50L1 tube
9. Stop Solution 50L1 tube
10. 3mol/L Sodium Acetate, pH 5.2 200L1 tube

Storage Condition 20
Product
Product Name Catalog No. Package Size
DsDD cDNA Subtraction Kit Wako 294-62001 for 5 reactions

Related Products
Product Name Catalog No. Package Size
Lambda Exonuclease 290-61501 1,000units

Exonuclease 294-61401 4,000units

dNTP Mixture 043-29291 0.2mL

Phenol/ Chloroform/ Isoamyl Alcohol (25 : 24 : 1) [NIPPON GENE CO., LTD.] 311-90151 250mL

052-07221 100mL
Ethanol (99.5)
054-07225 500mL

100bp DNA Step Ladder (100-1.5kbp) [Wako Chemicals USA] 546-01651 30g

Agarose S [NIPPON GENE CO., LTD.] 312-01193 100g

50TAE [NIPPON GENE CO., LTD.] 313-90035 500mL

6Loading Buffer Triple Dye [NIPPON GENE CO., LTD.] 314-90261 1mL3

Ethidium Bromide Solution [NIPPON GENE CO., LTD.] 315-90051 10mL

Distilled Water, Deionized, Sterile [NIPPON GENE CO., LTD.] 316-90101 100mL 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/

Start with an established cDNA Library


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. Wako Chemicals USA, Inc. Wako Chemicals GmbH
http://www.wako-chem.co.jp http://www.wakousa.com http://www.wako-chemicals.de

1-2, Doshomachi 3-Chome,


Head Office (Richmond, VA):
Toll-Free (U.S. only): 1-877-714-1920
European Office:
Nissanstrae 2, D-41468 Recovery of Intact Tester cDNA
Chuo-Ku, Osaka 540-8605, Japan Tel: 1-804-714-1920/Fax: 1-804-271-7791 Neuss, Germany
Tel: +81-6-6203-3741
Los Angeles Sales Office (Irvine, CA): Tel: 49-2131-311-0
Fax: +81-6-6201-5964
Tel: 1-949-679-1700/Fax: 1-949-679-1701 Fax: 49-2131-311100
Boston Sales Office (Cambridge, MA):
Online Cat.: http://search.wako-chem.com Tel: 1-617-354-6772/Fax: 1-617-354-6774

05803IBK
Worlds First New Subtraction Kit Using Enzyme Digestion Subtraction Efficiency [2]
12
The amplification levels of the highly expressed housekeeping genes
10
10.315 (GAPDH) and genes expressed differentially in hepatoma cells (AFP) were

(dierence in cycle number)


compared by real-time PCR. Subtraction efficiency was determined by the
From Physical Absorption Method to DsDD 8
Ct value obtained by subtracting the Ct value of HepG2 cDNA from the

Ct value
6
The subtraction hybridization method is a powerful technique used to identify genes that are specifically expressed in tissue, subtracted cDNA.
4
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
2 The subtracted cDNA of the highly expressed housekeeping GAPDH
0.26
several rounds of hybridization and took about 4 days. They also required the Tester and Driver cDNA to be prepared each time
0 genes dswere detected 10 cycles later than the HepG2 cDNA. The AFP
GAPDH AFP
2 genes, which are expressed specifically in HepG2, were detected after a
from mRNA because the cDNA Library could not be used as the starting material due to the influence of vector-derived sequences.
similar number of cycles. These results show that GAPDH is less than
Real-Time PCR Reaction Conditions 1/1,000 (1/210.315) and that highly efficient subtracted cDNA were
The DsDD cDNA Subtraction Kit Wako (patent pending) is a method based on
"Duplex-specific Direct Digestion" (DsDD), which overcomes traditional What is Subtraction? cDNA1ng/L
2Mastermix
5L
12.5L
50 2min.

constructed.
Example
subtraction methods. The Tester and Driver cDNA form drawbacks of hybrids of Tester Driver Sense Primer5M 1.5L 95 10min. Cycle Number Ct value
Antisense Primer5M 1.5L
genes, that are expressed nonspecifically. After hybridization, the hybrid ds Hepatoma cells / tissues Normal liver cells / tissues
HepG2 cDNA [A] Subtracted cDNA [B] [B][A]
SYBR Green I 0.75L 95 15sec.
cDNA from Tester and Driver cDNA are digested by duplex-specific A. Housekeeping genes A. Housekeeping genes dH2O 3.75L 60 1min.
nuclease. Finally, the Driver cDNA is removed by Exonuclease. This method B. Liver-specific genes B. Liver-specific genes Total 25L 40cycles
GAPDH 13.651 23.966 10.315
1 per minute from 60 to 95 AFP 17.884 17.624 0.26
efficiently enriches cDNA specifically expressed in Tester cDNA. C. Hepatoma-specific
For example, when analyzing specific functions and properties of cancer cell genes
tissue, the Tester is prepared from cancer tissue and the Driver from normal
(A,B,C)A,B) = C Principle
tissue, then cDNA derived from cancer specified gene is enriched. The DsDD
Subtraction is an efficient gene analysis method that
cDNA Subtraction Kit Wako is a revolutionary technique that uses the cDNA enriches the amount of differentially expressed genes Tester Library (abnormal) Driver Library (normal)
Library as starting material, and the subtracted Tester cDNA can be recovered by subtracting the expressed genes present in both A Tester cDNA Library and a Driver cDNA Library are
the Driver cDNA and the Tester cDNA. Target
prepared. In the cDNA Library for the Tester, the cDNA
intact. gene should be inserted in the same direction as the vector.

Features 1
Tester cDNA Amplification
Start with established cDNA Libraries During DNA amplification, a primer with phosphoric acid
added to the 5 end is used.
Intact recovery of the subtracted Tester cDNA DNA amplification
(such as PCR)
DNA amplification
(such as PCR)

Adoption of duplex-specific nuclease digestion


2
Lambda Exonuclease Treatment
Amplied Tester dsDNA from Library Amplied Driver dsDNA from Library The double-stranded DNAs 5 phosphorylated primer is
2 day performance 5
3
AAAAA
TTTTT
3
-P-5
5
3
AAAAA
TTTTT
3
5
recognized and is degraded by a 5 to a 3. Subtraction
Duplex-specific nuclease is purified from Kamchatka crab hepatopancreas efficiency is enhanced as there is no hybridization of

Target gene
5 AAAAA 3 5 AAAAA 3
3 TTTTT -P-5 3 TTTTT 5 Testers.
2 1 3

Subtraction Efficiency [1] Lambda Exonuclease


Treatment
Proper Restriction
Endonuclease Treatment
3
5 5
Restriction Endonuclease Treatment
cDNA Library from human hepatoma cell line HepG2 and human normal liver tissue are used as templates. The subtraction was AAAAA 3
3
AAAAA 3
The digestion of adapters on both ends ensures
3 TTTTT -P-5 TTTTT 5
performed using the DsDD cDNA Subtraction Kit Wako. The evaluation of the subtracted cDNA was conducted using highly 5 5
accurate hybridization results.
AAAAA 3 AAAAA 3
expressed housekeeping GAPDH genes and AFP genes, specifically expressed in HepG2 cells, as control. 3 TTTTT -P-5 3 TTTTT 5

PCR amplification of HepG2 cDNA (Tester ss cDNA), normal liver tissue cDNA (Driver ds cDNA), and subtracted cDNA was 4
1:200 Hybridization
performed, each sample being 1ng, and the results were analyzed by electrophoresis. The Tester cDNA and Driver cDNA react together at
After Hybridization , Duplex-specic
4
68 for 16 to 20 hours (mixing ratio = 1:200) Most of
nuclease Treatment5 the Tester cDNA form ds DNA due to the excessive
Tester Driver Tester-Driver amounts of Driver cDNA.
5 AAAAA 3 3 TTTTT 5
HepG2 cDNA Normal liver tissue cDNA Subtracted cDNA 3 TTTTT 5
5 AAAAA 3

Target gene
GAPDH AFP GAPDH AFP GAPDH AFP 5 AAAAA 3 5 AAAAA 3
5
3 TTTTT 5 Duplex-specific nuclease Treatment
5 TTTTT 3
3 AAAAA 5 5 AAAAA 3 Enzymes that specifically cleave ds DNA are used. A
3 TTTTT 5
high reaction temperature (68) makes reactions highly
1,500bp
After DNA amplication, specific. After the reaction, only the single-stranded
931 bp 1,000bp
859 bp Exonuclease Treatment6 DNA containing the Testers specifically expressed
5 5
genes will remain.
AAAAA 3 3 TTTTT
3 TTTTT 5
5 AAAAA 3
5
Target gene

AAAAA 3
AFP : alpha-fetoprotein 1% Agarose S 3 TTTTT 5 5 AAAAA 3 6
Exonuclease Treatment
GAPDH : glyceraldehyde-3-phosphate dehydrogenase 5 3
AAAAA
3 TTTTT 5 Enzymes that specifically cleave single-stranded DNA
3 TTTTT 5
are used. Non-specific genes are single stranded, thus
degraded.
The existence of highly expressed housekeeping GAPDH genes are confirmed by PCR amplification of HepG2 cDNA and 5 AAAAA 3
normal liver tissue cDNA, whereas not in subtracted cDNA. 3 TTTTT 5

Meanwhile, AFP genes, specifically expressed in HepG2 cells, were not confirmed with normal liver tissue cDNA, but in the 5 AAAAA 3
This highly efficient cDNA subtraction method
3 TTTTT 5
similar amplification level, the existence was confirmed with subtracted cDNA. gives results in only 2 days.
Subtracted cDNA
Worlds First New Subtraction Kit Using Enzyme Digestion Subtraction Efficiency [2]
12
The amplification levels of the highly expressed housekeeping genes
10
10.315 (GAPDH) and genes expressed differentially in hepatoma cells (AFP) were

(dierence in cycle number)


compared by real-time PCR. Subtraction efficiency was determined by the
From Physical Absorption Method to DsDD 8
Ct value obtained by subtracting the Ct value of HepG2 cDNA from the

Ct value
6
The subtraction hybridization method is a powerful technique used to identify genes that are specifically expressed in tissue, subtracted cDNA.
4
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
2 The subtracted cDNA of the highly expressed housekeeping GAPDH
0.26
several rounds of hybridization and took about 4 days. They also required the Tester and Driver cDNA to be prepared each time
0 genes dswere detected 10 cycles later than the HepG2 cDNA. The AFP
GAPDH AFP
2 genes, which are expressed specifically in HepG2, were detected after a
from mRNA because the cDNA Library could not be used as the starting material due to the influence of vector-derived sequences.
similar number of cycles. These results show that GAPDH is less than
Real-Time PCR Reaction Conditions 1/1,000 (1/210.315) and that highly efficient subtracted cDNA were
The DsDD cDNA Subtraction Kit Wako (patent pending) is a method based on
"Duplex-specific Direct Digestion" (DsDD), which overcomes traditional What is Subtraction? cDNA1ng/L
2Mastermix
5L
12.5L
50 2min.

constructed.
Example
subtraction methods. The Tester and Driver cDNA form drawbacks of hybrids of Tester Driver Sense Primer5M 1.5L 95 10min. Cycle Number Ct value
Antisense Primer5M 1.5L
genes, that are expressed nonspecifically. After hybridization, the hybrid ds Hepatoma cells / tissues Normal liver cells / tissues
HepG2 cDNA [A] Subtracted cDNA [B] [B][A]
SYBR Green I 0.75L 95 15sec.
cDNA from Tester and Driver cDNA are digested by duplex-specific A. Housekeeping genes A. Housekeeping genes dH2O 3.75L 60 1min.
nuclease. Finally, the Driver cDNA is removed by Exonuclease. This method B. Liver-specific genes B. Liver-specific genes Total 25L 40cycles
GAPDH 13.651 23.966 10.315
1 per minute from 60 to 95 AFP 17.884 17.624 0.26
efficiently enriches cDNA specifically expressed in Tester cDNA. C. Hepatoma-specific
For example, when analyzing specific functions and properties of cancer cell genes
tissue, the Tester is prepared from cancer tissue and the Driver from normal
(A,B,C)A,B) = C Principle
tissue, then cDNA derived from cancer specified gene is enriched. The DsDD
Subtraction is an efficient gene analysis method that
cDNA Subtraction Kit Wako is a revolutionary technique that uses the cDNA enriches the amount of differentially expressed genes Tester Library (abnormal) Driver Library (normal)
Library as starting material, and the subtracted Tester cDNA can be recovered by subtracting the expressed genes present in both A Tester cDNA Library and a Driver cDNA Library are
the Driver cDNA and the Tester cDNA. Target
prepared. In the cDNA Library for the Tester, the cDNA
intact. gene should be inserted in the same direction as the vector.

Features 1
Tester cDNA Amplification
Start with established cDNA Libraries During DNA amplification, a primer with phosphoric acid
added to the 5 end is used.
Intact recovery of the subtracted Tester cDNA DNA amplification
(such as PCR)
DNA amplification
(such as PCR)

Adoption of duplex-specific nuclease digestion


2
Lambda Exonuclease Treatment
Amplied Tester dsDNA from Library Amplied Driver dsDNA from Library The double-stranded DNAs 5 phosphorylated primer is
2 day performance 5
3
AAAAA
TTTTT
3
-P-5
5
3
AAAAA
TTTTT
3
5
recognized and is degraded by a 5 to a 3. Subtraction
Duplex-specific nuclease is purified from Kamchatka crab hepatopancreas efficiency is enhanced as there is no hybridization of

Target gene
5 AAAAA 3 5 AAAAA 3
3 TTTTT -P-5 3 TTTTT 5 Testers.
2 1 3

Subtraction Efficiency [1] Lambda Exonuclease


Treatment
Proper Restriction
Endonuclease Treatment
3
5 5
Restriction Endonuclease Treatment
cDNA Library from human hepatoma cell line HepG2 and human normal liver tissue are used as templates. The subtraction was AAAAA 3
3
AAAAA 3
The digestion of adapters on both ends ensures
3 TTTTT -P-5 TTTTT 5
performed using the DsDD cDNA Subtraction Kit Wako. The evaluation of the subtracted cDNA was conducted using highly 5 5
accurate hybridization results.
AAAAA 3 AAAAA 3
expressed housekeeping GAPDH genes and AFP genes, specifically expressed in HepG2 cells, as control. 3 TTTTT -P-5 3 TTTTT 5

PCR amplification of HepG2 cDNA (Tester ss cDNA), normal liver tissue cDNA (Driver ds cDNA), and subtracted cDNA was 4
1:200 Hybridization
performed, each sample being 1ng, and the results were analyzed by electrophoresis. The Tester cDNA and Driver cDNA react together at
After Hybridization , Duplex-specic
4
68 for 16 to 20 hours (mixing ratio = 1:200) Most of
nuclease Treatment5 the Tester cDNA form ds DNA due to the excessive
Tester Driver Tester-Driver amounts of Driver cDNA.
5 AAAAA 3 3 TTTTT 5
HepG2 cDNA Normal liver tissue cDNA Subtracted cDNA 3 TTTTT 5
5 AAAAA 3

Target gene
GAPDH AFP GAPDH AFP GAPDH AFP 5 AAAAA 3 5 AAAAA 3
5
3 TTTTT 5 Duplex-specific nuclease Treatment
5 TTTTT 3
3 AAAAA 5 5 AAAAA 3 Enzymes that specifically cleave ds DNA are used. A
3 TTTTT 5
high reaction temperature (68) makes reactions highly
1,500bp
After DNA amplication, specific. After the reaction, only the single-stranded
931 bp 1,000bp
859 bp Exonuclease Treatment6 DNA containing the Testers specifically expressed
5 5
genes will remain.
AAAAA 3 3 TTTTT
3 TTTTT 5
5 AAAAA 3
5
Target gene

AAAAA 3
AFP : alpha-fetoprotein 1% Agarose S 3 TTTTT 5 5 AAAAA 3 6
Exonuclease Treatment
GAPDH : glyceraldehyde-3-phosphate dehydrogenase 5 3
AAAAA
3 TTTTT 5 Enzymes that specifically cleave single-stranded DNA
3 TTTTT 5
are used. Non-specific genes are single stranded, thus
degraded.
The existence of highly expressed housekeeping GAPDH genes are confirmed by PCR amplification of HepG2 cDNA and 5 AAAAA 3
normal liver tissue cDNA, whereas not in subtracted cDNA. 3 TTTTT 5

Meanwhile, AFP genes, specifically expressed in HepG2 cells, were not confirmed with normal liver tissue cDNA, but in the 5 AAAAA 3
This highly efficient cDNA subtraction method
3 TTTTT 5
similar amplification level, the existence was confirmed with subtracted cDNA. gives results in only 2 days.
Subtracted cDNA
For Genetic Reseach

DsDD cDNA Subtraction Kit Wako


DsDD cDNA Subtraction Kit Wako
Kit Contents (for 5 reactions) Product Appearance
1. Lambda Exonuclease 5L1 tube
2. 10Lambda Exonuclease Buffer 25L1 tube
3. 4Hybridization Buffer 25L1 tube
Wako Catalog No. 294-62001
4. Duplex-specific nuclease 5L1 tube
5. 2Duplex-specific nuclease Buffer 25L1 tube
6. Exonuclease 5L1 tube
Simple and quick enrichment of specically expressed genes
7. 10Exonuclease Buffer 25L1 tube
8. Ethachinmate 50L1 tube
9. Stop Solution 50L1 tube
10. 3mol/L Sodium Acetate, pH 5.2 200L1 tube

Storage Condition 20
Product
Product Name Catalog No. Package Size
DsDD cDNA Subtraction Kit Wako 294-62001 for 5 reactions

Related Products
Product Name Catalog No. Package Size
Lambda Exonuclease 290-61501 1,000units

Exonuclease 294-61401 4,000units

dNTP Mixture 043-29291 0.2mL

Phenol/ Chloroform/ Isoamyl Alcohol (25 : 24 : 1) [NIPPON GENE CO., LTD.] 311-90151 250mL

052-07221 100mL
Ethanol (99.5)
054-07225 500mL

100bp DNA Step Ladder (100-1.5kbp) [Wako Chemicals USA] 546-01651 30g

Agarose S [NIPPON GENE CO., LTD.] 312-01193 100g

50TAE [NIPPON GENE CO., LTD.] 313-90035 500mL

6Loading Buffer Triple Dye [NIPPON GENE CO., LTD.] 314-90261 1mL3

Ethidium Bromide Solution [NIPPON GENE CO., LTD.] 315-90051 10mL

Distilled Water, Deionized, Sterile [NIPPON GENE CO., LTD.] 316-90101 100mL 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/

Start with an established cDNA Library


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. Wako Chemicals USA, Inc. Wako Chemicals GmbH
http://www.wako-chem.co.jp http://www.wakousa.com http://www.wako-chemicals.de

1-2, Doshomachi 3-Chome,


Head Office (Richmond, VA):
Toll-Free (U.S. only): 1-877-714-1920
European Office:
Nissanstrae 2, D-41468 Recovery of Intact Tester cDNA
Chuo-Ku, Osaka 540-8605, Japan Tel: 1-804-714-1920/Fax: 1-804-271-7791 Neuss, Germany
Tel: +81-6-6203-3741
Los Angeles Sales Office (Irvine, CA): Tel: 49-2131-311-0
Fax: +81-6-6201-5964
Tel: 1-949-679-1700/Fax: 1-949-679-1701 Fax: 49-2131-311100
Boston Sales Office (Cambridge, MA):
Online Cat.: http://search.wako-chem.com Tel: 1-617-354-6772/Fax: 1-617-354-6774

05803IBK