Indian Journal of Experimental Biology Vol. 44, October 2006, pp.

852-854

DNA isolation from goat blood using different brands of household detergents and its downstream application
O Suneel Kumar, M K Sharma & Dheer Singh*
Molecular Endocrinology Lab, Animal Biochemistry Division, National Dairy Research Institute, Karnal 132 001, India Received 13 November 2005; revised 17 July 2006 Rapid isolation of DNA from goat blood using different brands of detergents available in Indian market, is reported. The integrity and efficiency of these DNA preparations were compared with genomic DNA isolated by a standard kit (Flexi gene DNA kit), using amplification of exon 2 of CYP19 (aromatase) gene. The similar and significant amplification of this gene was obtained using genomic DNA isolated by kit and various detergents. However, among the detergents used, the Rin and Ezee were found to be the best to get DNA of high purity comparable to that obtained by kit. Keywor ds : Aromatase, Detergent, DNA isolation, PCR

Various methods have been described for the isolation of genomic DNA from tissue and peripheral blood. Some of these methods require large amount of samples and are not suitable for micro isolation. Further, these methods are little bit expensive and require specific reagents whose preparation is a time consuming process. In order to overcome these 1,2 problems, some workers have used laundry detergents for this purpose. A study on restriction fragment length polymorphism was made using genomic DNA from animal and plant tissues isolated with Persil 1 . However, a clear protocol and the comparative study on the usage of different brands of detergents for the isolation of genomic DNA are not available. Therefore, in the present investigation, a comparative study has been made on the isolation of genomic DNA from goat blood with different brands of detergents available in Indian market and Flexi gene DNA kit (QIAGEN, Cat# 51204) as the control. The integrity and efficiency of these DNA preparations were also compared for their downstream processing such as amplification of CYP19 (aromatase) gene.
_______________ *Correspondent authors E-mail: mks_scientist@yahoo.com E-mail: dheer_s2@rediffmail.com

Isolation of genomic DNA from blood Detergents used Different detergents graded by VOICE (Voluntary Organization in the Interest of 3 Consumers Education), New Delhi, India , such as Ariel (P&G), Tide(P&G), Active Wheel (Hindustan Lever Ltd, India), Henko stain Champion (Henkel, SPIC India Ltd), Rin (Hindustan Lever Ltd, India) and also Ezee (Godrej) liquid detergent were used. Detergent solutions were prepared in distilled water with a concentration of 20 mg/ml. RBC lysis buffer 0.3 M Sucrose (SRL), 0.01M Tris-HCl [ pH 7.5, SRL], 5 mM MgCl (Sigma), 1% 2 Triton X100 (Bio-Rad, USA). Collection of blood The blood from goat jugular vein was collected in UV irradiated tube containing EDTA (1 mg/ml). Primers for CYP19 (aromatase) exon 2 The primers were designed using Primer 3 software based on cattle sequence (Accession No. Z69242) and were as follows; Forward primer: 5'-GGGCTTGCTTGTTTTGACTC-3' Reverse primer: 5'-CCTGGTATTGAGGATGTGTCC-3' The protocol was followed according to Drabek and Petrek 2 with modifications. An aliquot of 5 ml of blood was taken in a 50 ml centrifuge tube. The RBC lysis buffer (40 ml) was added to this and the contents were centrifuged at 2700 g, at 15 o C for 5 min. The supernatant was discarded and centrifugation was repeated once again after adding 30 ml of lysis buffer and supernatant was discarded. The resultant WBC pellet was properly suspended in 1ml of 10 m M TrisHCl buffer ( pH 8) with the help of micropipette. The cell suspension so obtained was transferred to 2ml microcentrifuge tube and centrifugation was done at 675 g, at 4 o C for 2 min. The supernatant was discarded and cell pellet was again resuspended in 1340 l of 10mM Tris-HCl buffer ( p H 8). An aliquot of 670 l of cell suspension was transferred to another 2 ml microcentrifuge tube. The sterile glass bead (4m dia) and 670 l of detergent solution were added to each microcentrifuge tube containing 670 l cell suspension. The contents were mixed vigorously for 1 min to homogenize the cells. Then 500 l of NaCl was added to each tube and mixed vigorously for 30

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sec. The tubes were incubated at room temperature for 5 min and the contents were centrifuged at 12879g, at 4o C for 10 min to get rid of the cell debris. The supernatants so obtained were transferred to other microcentrifuge tubes and the centrifugation was repeated to remove any existing residue of proteins etc. The final supernatants of two tubes having same detergent, were pooled in 15 ml centrifuge tube. Absolute ethanol about 4 ml was added to pooled supernatant and mixed gently for precipitation of DNA. The DNA threads were retrieved by using heat sealed Pasteur pipette. The DNA on the Pasteur pipette was washed with 4 ml of 70% ethanol and immediately kept in 1 ml of 10 m M Tris-HCl buffer ( pH 8) for proper dissolution. Then, the DNA solution o was incubated at 60 C for 2 hr in a water bath to inactivate DNases. Finally, the DNA solution was stored at -20 o C until further use. Similarly, the two sets of WBC suspensions were processed for each detergent separately used for isolation of genomic DNA as per above protocol. Polymerase chain reaction (PCR) The PCR was performed in a total volume of 50 l of reaction mixture consisting of 1X PCR buffer [10 m M TrisHCl ( pH 9), 50 m M KCl, 1.5 m M MgCl 2 , 0.01% gelatin], 0.15 m M of each dNTP. 0.2 M of each primer and 1U Taq DNA polymerase. The amplification was done in a thermocycler using cycle conditions as 94 C for 2 min, 35 cycles of 94 C for 1 min, 60 C for 1 min, 72 C for 1 minute and a last cycle at 72 C for 5 min. Statistical analysis The data were analyzed by ANOVA, using MS EXCEL software. The genomic DNA was isolated from goat peripheral blood using different detergents as described. The purity factor (A /A 280 ) and yield of 260 DNA are presented in Table 1. The results showed a similar purity factor for DNA isolated by different detergents except the DNA isolated by Tide which exhibited a little impure preparation showing the purity factor (1.550.015) below the acceptable range (1.7-2.0). Among all the detergents used Ariel gave the DNA of highest purity (1.870.015) even better than that of control. However, the DNA yield with this detergent was very poor. Similarly, Active Wheel, Henko stain champion showed good purity factor but lower yield of DNA. Isolation of DNA using Rin and Ezee resulted in significantly higher purity factor and yield and these were comparable with that of DNA isolated by Flexi gene DNA kit.

The electrophoretic profile (0.8% agarose) of these DNA preparations obtained using all detergents has been shown in Fig. 1. The presence of single band with high intensity in all cases indicated the high integrity of DNA since there was no shearing of DNA observed on the profile. The DNA preparations obtained using all detergents and Flexi gene DNA kit were used for amplification of goat aromatase exon 2. The results (Fig. 2) of PCR product profile in 2% agarose gel indicated the presence of 352 bp band representing exon 2 in all DNA preparations. The results suggested that even the genomic DNA isolated by some of detergents showed poor purity factor and yield also, this did not affect exon 2 amplification. This further supports the view that these preparations had DNA of high integrity. The synthetic detergent powders consist of surface active agents (alkyl sulphates), builders and fillers. In addition, they have additives like antideposition agents, optical brighteners, bleaching agents, foam
Table1 Purity and yield of DNA isolated from goat blood using different brands of household detergents and Flexi gene DNA kit [Values are mean+ SE from 6 observations each] Detergent brand name Purity factor (A 260/A280) Yield (g/ml of blood)
a a

Ariel 1. 87 0.015 29.48 2.l2 Active wheel 1. 8l 0.022 28.34 1.34 Tide 1. 55 0.015 a 45.74 Henko stain Champion 1. 8l 0.016 28.68 2.57 Rin 1. 76 0.016 39.36 3.73 Ezee 1. 79 0. 017 38.69 1.95 Flexi gene DNA kit (Control) 1. 84 0.085 44.22 3.51
a , bindicate

1.5 b
a b b b

the significant difference. Critical difference for purity 0.01. Critical difference for yield 6.979

Fig. l Electrophoretic profile (0.8% agarose) of genomic DNA (100 ng) isolated from goat blood using different brands of detergents (Lane 1, Ariel; Lane 2, Active wheel ; Lane 3, Tide; Lane 4, Henko stain Champion ; [Lane 5, Rin; Lane 6, Ezee].

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INDIAN J EXP BIOL, OCTOBER 2006

Fig. 2 Electrophoretic profile (2% agarose) of amplified goat aromatase (exon 2) genomic DNA: The PCR was performed as per protocol described [Lane 1, 100 bp DNA ladder; Lane 2, 3,4,5,6,7 and 8 represent the PCR products after amplification of DNA isolated by Flexi gene DNA kit, Ariel, Active Wheel, Tide, Henko stain Champion, Rin and Ezee, respectively].

enzyme, may break down and denature the proteins. Addition of 5 M NaCI precipitates the denatured proteins due to salting out. In addition, alkyl sulphates 4 which are anionic surfactants may separate negatively charged DNA molecules from other molecules. The differences in the purity and yield of DNA between different detergents may be due to difference in the proportion of above compounds. Though it seems Rin and Ezee are best as far as purity and yield are concerned, there is no difference in downstream processing (PCR, the heart of molecular biology) among all detergents, suggesting that all the detergents used in present study can be recommended for DNA isolation and downstream applications such as PCR to small laboratories having budget constraints. This method is highly economic and efficient, because the use of proteinase K, phenol, guanidinium hydrochloride which is commonly used in standard DNA isolation protocols, are no more required. References
1 Bahl A & Pfenninger M, A rapid method of DNA isolation using laundry detergent, Nucleic Acids Res , 24 (1996) 1587. 2 Drabek J & Petrek M, A sugar laundry detergent and salt method for extraction of deoxyribonucleic acid from blood, Biomed Papers , 146 (2002) 37. 3 How green your detergent? Web: http://www.consumervoice.org/ 4 MSDS # FH/H/2002/GLGL-59YQJN, Tide granular Laundry Detergent MSDS.Doc.P&G.s

regulators, organic sequestering agents, enzymes (subtilisin, cellulase), perfumes, and substances to regulate density and assure crispiness of the clothes. During DNA isolation, by adding detergent solution the WBC will be lysed due to hyper osmosis and vigorous shaking. The detergent organic sequestering 4 agents and enzymes such as subtilisin , a proteolytic