Wako Pure Chemical Industries, Ltd. - Laboratory Chemicals / 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.

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.

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 (1/2^10.315) and that highly efficient subtracted cDNA were
constructed.

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.

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

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
Ethanol (99.5)	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.

	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