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World’s First New Subtraction Kit Using Enzyme Digestion
DsDD cDNA Subtraction Kit Wako
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.
Features
Start with established cDNA Libraries
Intact recovery of the subtracted Tester cDNA
Adoption of duplex-specific nuclease digestion
2 day performance
Subtraction Efficiency [1]
   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.
   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.
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
ΔCt value obtained by subtracting the Ct value of HepG2 cDNA from the
subtracted cDNA.

   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.
  Cycle Number ΔCt value
HepG2 cDNA [A] Subtracted cDNA [B] [B] - [A]
GAPDH 13.651 23.966 10.315
AFP 17.884 17.624 -0.26
Principle
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.

*1 Tester cDNA Amplification
     During DNA amplification, a primer with phosphoric acid
     added to the 5’ end is used.

*2 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 Restriction Endonuclease Treatment
     The digestion of adapters on both ends ensures
     accurate hybridization results.

*4 Hybridization
     The Tester cDNA and Driver cDNA react together at
     68°C 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.

*5 Duplex-specific nuclease Treatment
     Enzymes that specifically cleave ds DNA are used. A
     high reaction temperature (68°C) makes reactions highly
     specific. After the reaction, only the single-stranded
     DNA containing the Tester’s specifically expressed
     genes will remain.

*6 Exonuclease I 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.
Kit Contents (for 5 reactions)
1. Lambda Exonuclease 5μL×1 tube
2. 10×Lambda Exonuclease Buffer 25μL×1 tube
3. 4×Hybridization Buffer 25μL×1 tube
4. Duplex-specific nuclease 5μL×1 tube
5. 2×Duplex-specific nuclease Buffer 25μL×1 tube
6. Exonuclease I 5μL×1 tube
7. 10×Exonuclease I Buffer 25μL×1 tube
8. Ethachinmate 50μL×1 tube
9. Stop Solution 50μL×1 tube
10. 3mol/L Sodium Acetate, pH 5.2 200μL×1 tube
Storage Condition :-20°C
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 I 294-61401 4,000units
     dNTP Mixture 043-29291 0.2mL
     Phenol/ Chloroform/ Isoamyl Alcohol (25 : 24 : 1)    [NIPPON GENE CO., LTD.] 311-90151 250mL
     Ethanol (99.5) 052-07221 100mL
054-07225 500mL
     100bp DNA Step Ladder (100-1.5kbp)                       [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
Listed products are intended for laboratory research use only.
Please visit our online catalog to search for other products from Wako; http://www.e-reagent.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.