Indian Journal of Experimental Biology Vol, 39. September 2001. pp 906-9 I 0

Rapid extraction of DNA from diverse soils by guanidine thiocyanate method

Anupriya Agarwal*, Chitranshu Kumar & Reeta Goel Department of Microbiology, CBSH ,G.B. Pant University of Agriculture & Technology

Pantnagar 263 145, India

Received 25 May 2000; revised 29 March 2001

Molecular methods are being frequently used for the study of soi l microbial communities as majority of naturally occurring microbial populations are non-culturable. In the present study, we describe a protocol of DNA extrac tion from diverse soil s usi ng a combination of heat, enzy me (lysozyme) and guanidine thiocyanate . The efficacy of the procedure was evaluated in terms of yield. purity and duration of ex traction. The protocol was effective for neutral. acidic as well as alkaline soil s {pH range 4.5-8.5). The ex tracted soil DNA was observed with neg ligible shearing on agarose gel and the time taken for restriction digestion was very less. Further, the D A extracted was almost completely devoid of contaminants and pure enough which could be used for PCR amplification and Southern hybridi zation.

Major fraction of bacteria in soil (>99%) cannot be isolated on agar plates, and therefore no information about them can be obtained by conventional microbiological techniques. Recently, molecular methods have become valuable tools for the study of soil microbial communities. These methods depend upon efficient recovery of DNA from soil samples and thus obviate the need for cell culturing1.2. The DNA extracted, however contains large amount of humic material and is too impure for molecular analysis3

• The quality of extracted DNA has been improved by polyvinyl polypytTolidone treatments, CsCl-EtBr density gradient centrifugation and hydroxyapatite column chromatography4

. Modified extraction protocols have also been defined by several groups where detergent treatment and intensive mechanical disruption of the cell s are involved5

.15

• In other reports the mechani cal disruption of the cells are combined with other treatments such as hot or cold phenol

. t6 1. .d . I t7 - t<J f b .1 extractton , tqut mtrogen eye es , reeze- 01

cycles and microwave heating20. Several direct DNA

extraction procedures from soi l do not use drastic disintegration step, but rather employ lysis of soil microorganisms by sodium dodecyl sulphate (SDS)21

,

a combination of SDS and heat treatments2 or sarcosyl and heat treatments 22

.

Objective of this study is to develop a rapid and universal D A extraction protocol for soi ls of diverse

* Present add ress: Petroleum Toxicology Division. Industri al Tox icology Research Centre, P.O. Box-80. M.G. Marg, Lucknow -226 00 I. India Fax : +91-0522-228227 E-mail : nupri yaagarwal@usa.net

composition. This DNA extraction protocol is a modification of the method reported by Porteous et a/. 1 in which a combination of heat, enzyme and guanidine isothiocyanate were employed. In the present study guanidine thiocyanate which is an inorganic compound, used for R A isolati01P and genomic DNA extraction from aquatic sources24 was used to extract DNA from soil.

Materials and Methods

Environmental samples- Five soil samples of varying pH (pH 4.5-8.5) were collected from Durgapur, Dehradun, Ujhani, Allahabad and Pantnagar. Local Pantnagar soil , used for standardization of the protocol was clay loam in texture containing 20-25% sand, 45-50% silt and 25-35% clay. The pH of the soil was 7.2 . Sterilized soil was used for the determination of extraction efficiency. For sterilization, soil was sieved through a 2 mm mesh and autoclaved twice at 12 1 °C for lhr at 15 lb/in2 on altemate days. All soil samples were stored in plastic bags at 4°C.

Soil samples analysis- Percent C and N, organic content and pH of the soil samples were determined by standard methods 25 (Table 2).

Bacterial strains~The microorganisms used for testing the efficiency of the extraction procedure were Pseudomonas jluorescens (MTCC 13525 procured from culture collection at lMTEC, Chandigarh), Rhizobiu111 sp., E. coli, Bacillus sp., Pseudomonas j luorescens PRS9

& GRP3 (isolated from the rhizoplane and rhizosphere of various legumes) obtai ned from departmental

AGARWAL eta/ . :RAPID DNA EXTRACTION BY USING GUANIDINE THlOCYANATE 907

culture collection. All cultures were stored at 4°C on respective agar slants and at -20 ·c in 50% glycerol stocks.

Detennination of cfit-Sterilized Pantnagar soil was seeded with bacterial culture of known cell density ( -109 cells/ml). Pseudomonas fluorescens strain PRS9 was used as seed organism and I ml of cell suspension was mixed with I 0 g of sterilized soil. Serial dilutions of seeded soil was prepared and microbial population was determined as colony forming units (cfu/g) of soil by plating I ml of each dilution on sodium succinate agar plates and incubating at 37°C overnight.

DNA isolation-The protocol was developed as follows: I g (wet weight) of soil sample was mixed with

3.5 ml of homogenization buffer [2.5 M NaCl and 0.25 M Na2EDTA (pH 8.0)] and vortexed for 30 sin a 10 ml centrifuge tube. Homogenate was then sonicated (Labsonic U.B. Braun) at room temperature for 3 min, using repeating duty cycle of 0.3s. 1 mg lysozyme (Sigma Chemical Co.) was added, vortexed for 10 sand the sample was incubated at 37°C for I hr. Lysis solution (3.5 ml) containing 250 mM NaCI, 100 mM

Na2EDT A, (pH 8.0), 4% SDS and 500 jll of 8 M guanidine thiocyanate (SRL Pvt. Ltd., Bombay) was added to the above mixture and contents were mixed by vortexing for 10 s. Tubes were incubated at 68°C for I hr in a water bath and centrifuged twice at 12,000 g for 15 min at 4°C (Remi C-24 centrifuge). The supernatant was extracted with an equal volume of phenol equilibrated to pH 8.0 with 0.1 M Tris-HCI and centrifuged at 5,500 g for 10 min. The aqueous phase was rextracted once with phenol:chloroform (I: I) and once with chloroform: isoamylalcohol (24: I). Supernatant was mixed with 0.6 volume of isopropanol and kept at -20°C for I hr followed by centrifugation at 12,000 g for 15 min. Pellet obtained was washed with 70% ethanol, dried by aspiration and resuspended in TE buffer (I 0 mM Tris-HCI, I.OmM EDT A, pH 8.0). Crude DNA was further purified through Qiagen gel extraction kit QlAEX II and Sephadex G-200 column separatell6

.

This protocol was also tested for soils seeded with different bacterial strains and unsterilize diverse soil samples. Genomic DNA from pure cultures was extracted by following the protocol of Sambrook et aP3

.

Restriction digestion--Restriction digestion of DNA extracted from pure culture and soil DNA sample were carried out for I hr and 4hr respectively at 3iC. 20 jll of reaction mixture contained I x restriction enzyme buffer, 40 units of Bgl II

(Boehringer Mannheim, GmbH Germany) and 1.0-1 .5

jlg DNA.

Gel electrophoresis and quantification--Purified genomic DNA, extracted from soil sample and pure culture (intact and after restriction digestion) were electrophoresed at 50 V for 2 hr with I X T AE in I%

agrose (Sigma Co.) along with Hind III digested A DNA as molecular weight marker. Gel was stained for

15 min in IX T AE containing ethidium bromide (0.5 !l g/ml). Bands were visualized on a U.V. Transilluminater (Fotodyne. Inc., New Berlin, Wis.) and photographed. The DNA in the sample was quantified by comparing the fluorescence intensities of ethidium bromide stained DNA bands with that of standard molecular weight marker by using the method of Zhou et al 2

• Purity was checked by measuring the A2&;/A280 ratio in U.V. spectrophotometer (Beckmann. DU). Percent efficiency was determined by dividing experimental DNA yield with literature value reported for cellular DNA content of soil bacteria (1.6 fg /cell) as reported by Bakken and Olsen 27 and multiplying with 100.

Results and Discussion

Genomic DNA extracted from soil sample was visualized on an ethidium bromide stained 1% agarose gel (Fig. I).

The efficiency of recovery of DNA by this protocol in sterile seeded soil having approximately 2± 0.03 x 108 cfu/g , is 93% (Table. I) which is comparable to the earlier reports being 92 to 99% in SDS based extraction method 16

·17

, 90% lysis efficiency by bead mill homogenization3

, 95% by EDT A and lysozyme treatment with three freeze thaw cycles18

·20

.

This protocol involved initial homogenization of the samples in buffered salt solution, followed by vigorous vortexing and sonication. This method was employed previously in our laboratory for protein extraction which resulted in complete lysis of bacterial cells. This indicates that sonication could be effective not only in extracting DNA but also in releasing protected bacteria making them available for subsequent lytic treatments. This treatment also separates the soil debris and fungal mycelia from bacterial cells. Lysozyme, guanidine thiocyanate and heat treatment were used to lyse bacterial cells for releasing DNA. Guanidine thiocyanate was helpful for disruption and separation of cells as well as for protein denaturation. It was also used to protect free DNA from high levels of endogenous nucleases. Moreover, RNA contamination was not observed

908 INDIAN J EXP BIOL, SEPTEMB ER, 200 1

(Fig. I) which indicates RNA degradation had occurred dUiing the cell lys is thereby it was obviating the need for RNase treatment. Increase in the concentration of molarities of homogenization buffer components and guanidine thiocyanate were significant because these chemicals not only enhance lysis efficiency of bacterial cell s but they also separate DNA from contaminants such as humic acid, polysaccharides, proteins, tannin etc. that inhibit restriction digestion. There was no adverse effect of increased concentration of these chemicals on restnct1on digestion. Minimal centrifugation steps were used to separate DNA from cell debris and contaminating particles, thereby minimizing the sheming of DNA and shortening the total extraction time. DNA can be extracted from Jm·ge

( Kb)

2 3 .o

2.3

Fig. 1- Genomic D A extracted from soi l and pure culture. M: Hind Ill d iges ted lambda DNA Marker Lane I : Intact genomic soil DNA. Lane 2: So il DNA di gested with Bgl II (4hr). Lane 3: Intact genomic pure cu lture DNA. Lane 4 : Pure c ultu re DNA digested with Bgl II ( I hr)

number of samples in 8 hr which is comparable with Porteous et al. 1• Porteous et al. observed sheared DNA occasionally appeming a large smear but by the present method we observed negligible shearing.

Two purification methods were compared in terms of purity a11d recovery (Tablel ). Gel purification from QIAEX IJ Gel Extraction Kit was based on differential electrophoretic mobilities of DNA and humic materials. Pmification by Sephadex G-200 spin column was based on pore matrix of binding resin. Sephadex G-200 column purification method resulted in higher level of DNA recovery i.e. 83-85% which is comparable with Mil ler et a/ 28

. It is also better for removal of inhibitors

and thi s column could be reused (stored at 4°C) after washing with TE buffer. Pmity and quality of DNA purified by both methods were same.

The method described here is simple, economic, precise and will facilitate the analys is of Im·ge number of environmental samples containing di fferent bacterial strai ns incl uding Pseudo111onas jluorescens, E. coli, Rhizobium sp., Bacillus sp.

Further, the protocol was also tested on acidic as well

alkaline soils (pH range 4.5-8.5) and was found to be adequately effective (Table 2). The puri fied DN A

extracted from thi s protocol was good enough to be

utilized for restriction diges tion (Fig. l ). The time

taken for restriction digestion of soi l DNA was more as compared to pure culture DN A, indicating the

recovered DNA cou ld have some inhib itors but the

time taken fo r restri ct ion digestion of so il D A was

however very less when compared to studies reported

by Porteous et a /1• Studies are in progress to utilize

this rapid DNA extract ion method fo r other molecular

biology techniques such as PCR amp lification and Southern hybridi zation.

Table !-Compari son of DNA ex tracted from soi l sample in terms of DNA yie ld, ex tract ion effi cie ncy and pu rity subjected to two different treatments

Sample DNA yielda Puritl % Recovery

Crude so il DNA

Purified by QIA EX II Gel ex traction kit

Purified by Sephadex G-200 spin column

(~tg/g wet wt of soil )

0.300±0.03

0.057 ± 0.003

0.253 ± 0.002

1"1DNA yields were estimated by gel sta ining (mean va lue ± SO with n = 3)

(b) Determined by U. V. sphectrophotometer as A 2601280

A 2601280

1.20 ± 0. 10

1.54 ± 0.05

1.54 ± 0 .05

93%c

18-20%d

83-85%d

col Percent e fficiency was determined by di viding experimental DNA yie ld (DNA y ield by ge l stain ing) with li terature value reported for cellular DNA content of so il bacteria ( 1.6 fg/ce ll as reported by Bakken &Olsen27

) and multi plying with 100

cdJ Percent recovery o f purificatio n was determined by taking crude DNA yie ld as 100%

AGARWAL e1 a/. : RAPID DNA EXTRACTION BY USING GUANIDINE THIOCYANATE 909

T able 2 - DNA recovery from soil s of diverse co mposition

So il sample pl-1" %C Organic % N DNA yie ldb location contelll ().lg/g wet wt of soil )

Dehradun 4 .5 0. 39% 0.68% 0.04% 2.0±0.05

Durgapur 7.0 0 .74% 1.28% 0.1 % 1.9 ± 0.05

Pantnagar 7.2 1.23% 2. 12 % 0 .64%c 1.1 3 ± 0. 18

Ujhani 8.0 1.58% 2.7% 0.1 3% 0.6±0.08

Allahabad 8.5 1.97% 3.4% 0 .20% 3. 1 ±0. 14

1''

1Measured after equilibra ted with water 1"lDNA yields from indegenous mi cronora preselll in unsterile so il , yie lds were estimated by gel staining

(mean value± SO with n = 3) kl T he sample was taken from the restri cted area which was regularl y under the treatment o f bio logical manure

Acknowledgement This work was funded by UGC and Department of

Biotechnology, Indi a. Authors, AA and CK also acknowl edge graduate research ass istantship obtained during the course of this study.

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