VEGETOS
Vol. 25 (2) : 13-19 (2012)
An Efficient and Rapid Method of DNA Extraction For Molecular Marker Studies in Cotton (Gossypium hirsutum L.) Shiv K. Yadav*, V. Vashisht, S.S. Gaurav, H. Sindhuja, D.V. Singh and S. K. Lal Division of Seed Science and Technology, Indian Agricultural Research Institute, New Delhi-110012.
The plant tissues with rigid cell wall need to be ground in liquid nitrogen for disruption of cells. Further, they are transferred to an extraction buffer containing detergents as well as reducing and chelating agents to disrupt the membranes. However, it can cause serious degradation of the genomic DNA. Moreover, the oil and phenolic compounds in cotton seeds degrade the quality and quantity of extracted DNA. The quality and purity of DNA is essential for the success of the molecular studies. Hence, it was considered worthwhile to evaluate various methods of DNA isolation in terms of DNA yield and amplification. In these studies, three methods (Mini Prep, CTAB and G-Bioscience DNA isolation Kit) were used to isolate DNA from five transgenics (Bt.) cotton hybrids. The results revealed that the highest quantity of DNA was extracted with the Kit method, followed by CTAB and Mini prep method respectively. Though the DNA isolated with Mini Prep method was quantitatively less but it was comparable with Kit and CTAB methods with respect to quality. Hence, Mini Prep method can be utilized effectively for screening of genotypes for genetic purity assessment by PCR based markers. Keywords: DNA extraction methods, Cotton, Genetic purity, molecular markers. Received: November 08, 2011 Revised: June 15, 2012 Accepted: November 16, 2012
INTRODUCTION Cotton is one of the important cash crops and the most important raw material for the textile industry. Cotton production is considered to have a wide reaching impact not only on the livelihood of farmers and economy of country, but also on the international trade. The cotton production has taken a quantum jump mainly attributed to the increased area under a number of Bt. hybrids, containing a single or stacked combination of events, approved for cultivation in various parts of the world. The hybrid seeds of Bt. cotton are sold in packets containing 450 gm of Bt. and 120 gm of non-Bt. Seed as required for refuge, sufficient enough to plant one acre. In 2002, the year of introduction, a packet containing 450 gm of Bt. cotton hybrid seeds was being sold at quite high price, but with governments stepping in for controlling the price of Bt. cotton hybrids, a number of companies have reduced the pric* Corresponding author Email :
[email protected]
es substantially. Reduced prices have meant that while farmers were initially planting only one packet of seeds for every acre, they have to now raise it to 1.2 to 1.5 packets. Thus, cotton hybrid seed is a high value commodity. This warrants our attention to assure high quality of such high value input (i.e. cotton seed), servicing the cotton industry. The genetic purity testing is more important in cotton hybrids, which are mostly converted as Bt. cotton hybrids by the respective seed companies. Kranthi et al. (2005) reported a variation in toxin expression of Bt. gene due to the change in parental background of the hybrids. Therefore, maintenance of high level of genetic purity of Bt. hybrid is essential to exploit the level of heterosis and extent of protection observed in cotton crop. This demands a strict vigil on the genetic purity standards of a commercial Bt. cotton hybrid seed lots. 13
Shiv K Yadav et al. DNA molecular marker technologies such as RAPD (Williams et al. 1990), CAPS (Konieczny and Ausubel, 1993), STSs (Mazur et al. 1995), microsatellite (Panaud et al. 1996), and AFLP (Vos et al. 1995), have been used in plants for molecular mapping, identification of genotypes, characters associated with genes of interest and genetic diversity studies. The PCR-based markers being the most popular and efficient, has been advancing rapidly during past decades and its application perspective seems very brilliant in crop breeding, varietal purity testing of crop seeds and germplasm fingerprinting (Fang et al. 2000). Meanwhile, PCR is also an essential approach for detection of target genes in transgenic breeding and genetically modified (GM) crops. The DNA extraction method was first established by Marmur in 1961 using SDS and chloroform. Since then, scientists have been constantly devoting themselves to the improvement and simplification of DNA extraction methods and many kits for DNA isolation are now commercially available in the market. However, these commercial DNA kits are very reliable and user-friendly, but too expensive to be the choice of researchers working on applied sciences, e.g., seed technology in developing countries. On the other hand, most improved or simplified DNA protocols are still very tedious and time-consuming when applied for large samples in seed testing programs. Therefore, DNA extraction has become a major bottleneck for large-scale applications of DNA markers and transgenic detection. Several methods for minimizing the DNA extraction steps have been reported (Bourquin et al. 1991), but they require comparatively more plant tissue to be grounded in liquid nitrogen. The grinding of plant tissues with rigid cell wall in liquid nitrogen for disruption of cells and then transferring to an extraction buffer that contains detergent to disrupt the membranes and the reducing and chelating agents to inactivate nucleases is the protocol routinely followed. This can cause serious degradation of the genomic DNA, moreover,
Fig 1. Stage of seedlings at which leaves from plants grown in glasshouse were taken
Seedlings were grown in a mixture of soil and compost (3:1), sterilized at 1500C overnight in glasshouse at 250C after surface sterilization of seeds with 1% mercuric chloride (HgCl2). Pooled leaf tissues from partially expanded leaves of 10 plants were collected from approximately 20 days old seedling (Figure 1) for extraction of DNA to be used for evaluation of the parental lines and hybrids. the oil and phenolic compounds in cotton distracts the quality and quantity of extracted DNA. Additionally growing plants and storing samples in freezers is often difficult due to space constraints. To overcome such problems, Chunwongse et al. (1993) developed a DNA extraction method using the dry half seeds of rice and wheat. However, the method has not been used extensively for other plant seeds, especially the oil containing seeds like cotton. Thus, a simple, rapid and comparable DNA extraction method is needed for analysis involving large number of samples.
Stock solution
Working solution
For 100 ml extraction buffer
1 M trisHCl (pH 7.5): 121.14 gm Tris in litre of double distilled water 5 M NaCl : 58.44 gm of NaCl dissolved in 1 litre of double distilled water 0.5 M EDTA (pH 8.0):372.24 dissolved in 1 litre of double distilled water
100 mM Tris
10 ml from stock
1.5 M NaCl
30 ml from stock
20 mM EDTA
4 ml from stock
iv.
2% CTAB
2 gm
v.
0.2% 2 β-Mercaptoethanol
200 µl
vi.
2% PVP
2 gm
i. ii. iii.
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An Efficient and Rapid Method of DNA Extraction
MATERIALS AND METHODS Materials i. Seeds Pure seeds of 5 Bt. cotton hybrids namely Arjun, Cotton Express, NCH 31, NCH 34 and Vishwanath along with their parental lines were obtained from M/s Nath Seeds, Aurangabad and used in the present study for isolation of genomic DNA suitable for molecular marker profiling. All analyses were made on pooled seedling samples, except for genetic purity testing where single seedling analysis was done. ii. Chemicals All chemicals used in the present study were of analytical/molecular grade, unless otherwise stated. Methods Three methods namely CTAB, DNA isolation kit (OmniPrepTM from G-Biosciences) and Mini prep were evaluated for isolation of genomic DNA for comparing the quantity and suitability of extracted DNA for its use in molecular marker analysis. I. CTAB method All the reagents used for the DNA extraction are given as below: i. Preparation of buffers and solutions for DNA extraction a) Preparation of DNA extraction buffer The final volume of the buffer was made upto 100 ml with autoclaved double distilled water. The buffer was kept in hot water bath at 650C to completely dissolve the CTAB. βExtraction Buffer Tris HCl (pH = 8.0)
50Mm
EDTA
25Mm
NaCl
300Mm
SDS
1%
Mercaptoethanol was added to the extraction buffer immediately before use. b) Chloroform: Isoamyl alcohol Chloroform and isoamyl alcohol were mixed in 24:1(v/v) ratio. c) 70% Ethanol 70 ml of ethanol was mixed with 30 ml of double distilled water. d) Tris-EDTA buffer TE buffer (10 mM tris, 1 mM EDTA pH 8.) was prepared by mixing 1 ml of 1 M tris (pH 8.0), 200 µl of 0.5 M EDTA (pH 8.0) and the final volume was made upto 100 ml. using double distilled water and was finally autoclaved. e) Preparation of RNAase 4 mg of RNAase was dissolved in 2 ml of autoclaved double distilled water and was stored at -200C. f) Preparation of electrode buffer (Tris-Glacial acetic acid, EDTA (TAE) Buffer) TAE (50x) stock buffer (pH 8.0) was prepared by dissolving Tris-base (242.0 g), Glacial Acetic acid (57.1 g), EDTA (46.531g, pH 8) and the final volume was made upto 250 ml with double distilled water. From this 50 x TBE stock, working buffer of 1 X TAE was prepared by mixing 10 ml of stock buffer with 490 ml of double distilled water to get a final volume of 1 litre. Approximately 500 ml of this 1 x TAE was poured into the electrophoresis tank, so as to keep the gel immersed in it. g) Ethidium bromide 100 mg Ethidium bromide was dissolved in 10 ml double distilled water. h) Preparation of Lamda DNA (10 mg/µl) From the stock of Lamda DNA (10 ng/ml), 2 µl DNA was taken and dissolved in 58 µl TE to get 10 ng/µl working solution. ii Isolation of DNA Two grams of fresh young leaves of parental lines and hybrids were collected in sterile polythene bag, frozen in liquid nitrogen and used for DNA isolation as described by Edward et al. (1991).
Table 1. Details of the sequences of the RAPD primers Name of the primers
Sequences of the primers
Name of the primers OPA-13
OPA-07
5'-GAAACGGGTG-3'
OPA-08
5'-GTGACGTAGG-3'
OPA-15
OPA-10
5'-GTGATCGCAG-3'
OPB-02
OPA-11
5'- CAATCGCCGT-3'
OPB-04
Sequences of the primers
5’-CAGCACCCAC-3’ 5'-TTCCGAACCC-3' 5'-TGATCCCTGG-3' 5'-GGACTGGAGT-3' 15
Shiv K. Yadav et al. Fig 2. 34, 3. NCH 31, 4. Nathbaba, 5. Arjun and 6 Cotton Express)
Fig 3. Single seedling analysis of Vishwanath hybrid with OPA-7 RAPD primer M
H
H
H
H
.II. KIT method: The OmniPrepTM kit (Cat.# 786-136) from GBiosciences containing; Genomic Lysis Buffer (100ml), Nuclei Isolation Buffer (2X30ml), DNA Stripping Solution (10ml), Precipitation Solution (20ml), Mussel Glycogen (10mg/ml) (1ml), TE Buffer (20ml), RNase (LonglifeTM) (0.5ml) and Proteinase K (LonglifeTM) (2X0.5ml) was used to isolate DNA from the plant tissue. For isolation of DNA, the steps mentioned in the brochure of the kit were strictly followed. Rest of the buffers and solutions for DNA extraction were prepared as explained above in CTAB method. A. DNA isolation A simple and efficient protocol for extraction of high quality genomic DNA from young leaves of cotton was de-
H
H
H
H
F
veloped with some modifications in CTAB method. The important steps in this protocol include; Approximately one gram (0.8g) of fresh leaves from 20 day old seedlings was taken. 900µl (300+300+300) extraction buffer was added to grind the sample. The sample mass was transferred to 1.5ml micro centrifuge tube. 0.5-1ml chloroform: isoamyl alcohol (24:1) was added, mixed well for 20 minutes and centrifuged at 12,000 rpm for 10 minutes. The top aqueous phase containing the DNA was removed carefully and transferred to a new tube. One volume (200-500µl) of 3M sodium acetate (pH 5.2) was added to the solution and mixed well. 16
An Efficient and Rapid Method of DNA Extraction 2-2.5 volume of ice-cold 100% ethanol was added, mixed well and placed in ice for 20 minutes. It was centrifuged again for 8 minutes at 10,000 rpm and the supernatant was removed. 1ml ethanol (70%) was added and the tube was inverted several times followed by centrifugation for 2-3 minutes (10,000 rpm).This step can be repeated (3-4 times) as per the impurities observed in the extracted DNA. The supernatant was removed and the pellets were dried. The dry DNA pellet was dissolved in TE (pH 8.0) and stored at 4°C. B. Purification of DNA Purification of DNA is required to remove RNA, protein, polyphenols and polysaccharides, which are considered as major contaminants in DNA precipitates. RNA was removed by RNAase treatment and proteins were removed by phenol chloroform extraction. 1 µl RNAase was added to DNA sample (1 µl/100 µl DNA) and kept at 37°C for 1 hr. Equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added to the Eppendorf tube containing DNA sample. The tube was spun at 10,000 rpm for 5 minutes at room temperature. The aqueous phase was taken and transferred to fresh Eppendorf tube. Equal volume of Choloform: Isoamyl alcohol (24:1) was added and mixed gently for 5 minutes. It was centrifuged at 10,000 rpm for 5 minutes and the aqueous phase was collected. 3M sodium acetate was added to the volume of aqueous phase, mixed properly and kept for 10 minutes. Two volume of chilled absolute alcohol was added, mixed properly and kept at room temperature for 1 hr. Subsequently, it was spun at 10,000 rpm for 5 minutes at 4°C. The supernatant was discarded and the pellet was collected and washed twice with 70% ethanol. The pellet was dissolved in TE buffer at 40C and DNA was stored at -200C. C. Checking the quality and quantity An agarose gel (0.8%) was prepared by solublizing 0.8 gm of agarose in 100 ml of 1 x TAE in a microwave oven till agarose was dissolved and cooled to room temperature and finally, Ethidium bromide @ 50 µg/ml was added. Later, it was poured into the gel casting tray in which the combs were set. After one hour, the gel was solidified and the combs were removed to form the wells. To 4 µl of DNA sample, 2.0 µl of dye was mixed and loaded into the wells. Gene ruler DNA ladder plus was loaded as control in the corner well (3 µl of marker DNA ladder plus 2 µl of dye). The gel was run at 50V for 1 hr. The quality of DNA was judged by the nature of the band at the corresponding position vis-avis control. The presence of a single compact band indicated
that isolated DNA was of high molecular weight and good quality. The approximate quantity of the DNA in the sample was estimated in a Spectrophotometer (Beckman 640 DU) by taking absorbance ratio at wavelengths of 280 and 260 λ. After quantifying the DNA of each genotype, the samples were diluted with TE buffer so that final concentration of DNA was ~ 10 ng/µl. D. Using DNA extracted with mini prep method for testing hybridity by RAPD markers Among the DNA marker techniques used for variety identification and verification, RAPD is technically the simplest, least expensive, fast and does not require huge infrastructure to start with (Welsh and McClelland, 1990). Hence, DNA extracted by Mini Prep method was used for genetic purity analysis in Vishwanath hybrid with RAPD markers. Ten random decamer primers from the operon series were screened on template DNA from parental lines and hybrid under study. Three oligonucleotide primers, giving good scorable amplification products, were selected for further RAPD analysis. The details regarding the sequences of the primers are provided below (Table-1). RAPD analysis RAPD analysis was carried out using standard PCR (Polymerase Chain Reaction) to selectively amplify a specific segment of the total genomic DNA in vitro mani fold (Mullies et al., 1986) using the primers listed. The primers whose details are given in table 1 were used for RAPD analysis. The PCR amplification was performed as described by Williams et al. (1990) in a 0.2 ml reaction tube with total volume of 25 µl containing 25 ng of template DNA 0.2 µm primer, 400 µm dNTP mix (MBI Fermentas), 1.5 mM/µl MgCl2, 1 X PCR assay buffer (Bangalore Genei, India) and 1 unit Taq DNA polymerase (Bangalore Genei, India). The PCR was performed as follows: Initial denaturation 940C for 5 min Cycle denaturation 940C for 1 min Annealing 350C for 1 min Extension 720C for 2 min Final extension 720C for 8 min The PCR reaction was carried out for 40 cycles. The final product was electrophorised on 1.5% agarose gel alongwith 1 kb. DNA ladder (MBI, Fermentas) as a marker in 1 x TAE buffer at 60 V for 3-4 hrs. The gel was stained with Ethidium bromide and photographed using the gel documentation system. Each RAPD analysis was reconfirmed by at least two replications. The amplified products were scored separately for each primer. The bands observed on the gels were compared across the lanes for products with similar molecular weights.
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Shiv K. Yadav et al.
RESULTS AND DISCUSSION Testing of genetic purity of Bt. hybrid cotton seed is mandatory prior to its sale and distribution. The quality of DNA and rapidness in extraction is crucial for the success in results of genetic purity testing by molecular markers at commercial level. Hence, three methods (Mini Prep, CTAB and G -Bioscience DNA isolation Kit) were used to isolate DNA from transgenics (Bt.) cotton hybrids and their parental lines with the main goal to evaluate various methods of DNA isolation in terms of DNA yield, speed with economy in extraction and amplification quality in this study. The isolated DNA was quantified with Bechman DU and the highest quantity of DNA was obtained from 2 grams of partially expanded leaves in the Kit method (2579 µg), followed by CTAB (2100 µg) and Mini prep method (879.2 µg) respectively (Table 2). These results are in conformity with those reported by Mauro et al. (1992), in which the best results were obtained from rapidly expanding leaves, one to two nodes from the shoot tip. The DNA yields from fresh leaf tissues of Bt. cotton by the above mentioned procedures ranged from 2639.5µg in Cotton Express genotype (Kit method) to 746.5µg in Nathbaba (Mini Prep method) observed at 260 OD. The DNA yields obtained with Mini Prep method was highest in Cotton Express (1007.1). However, DNA quantity in all hybrids was good enough for conducting molecular marker analysis. It was found that DNA extracted by Mini Prep method was occasionally brownish in color and required 4-5 washings with 70% Ethanol. The inclusion of CTAB in DNA extraction buffer helps in elimination of polysaccharides for improving the quality of DNA. NaCl has been used to remove polysaccharides (Fang et al., 1992) and PVP to purge polyphenols (Maliyakal, 1992). The quality of DNA isolated with Mini Prep method was comparable with Kit and CTAB methods for its use in screening of PCR based markers (Figure 2) where, RNA was removed by RNAase treatment and proteins were removed by phenol chloroform extraction. The simplicity and speed of the Mini Prep procedure makes it very practical and rapid so that 30 - 40 DNA samples can be processed in a single day. Even the minimum DNA isolated with Mini Prep method in Vishwanath hybrid was found to be amplifiable in PCR using the RAPD technique (Williams et al. 1990). This suggests that the DNA thus obtained was of good quality. Webb and Knapp (1990) and Fang et al. (1992) also suggested that the DNA amplification is possible only in absence of contaminants. Polysaccharides are difficult to separate from DNA (Murray and Thompson, 1980). Polysaccharides interfere with several biological enzymes such as polymerases, ligases and restriction endonucleases (Shioda et al. 1987; Richards, 1988). However, complete amplification in PCR indicated the absence of polysaccharides in DNA isolated
with Mini Prep method. Each of the individual primers produced 3 to 15 amplified products with a size variation from 500 bp to 1000 bp. However, only 2-4 fragments were of high intensity whereas the intensity of other fragments was clear, but low. A total of 141 amplification products were generated from 10 primers. Among the polymorphic primers, reproducibility of OPA-07 was the highest. Hence, it was selected for hybrid purity testing and detection of selfed seed in the commercial seed lots of hybrid Vishwanath. Primer OPA-07 was found to generate most polymorphic fragments in case of hybrid Vishwanath which could facilitate the differentiation of the hybrid from the female parent (Figure 3) In addition to OPA-07, OPA-13 and OPA-08 were also polymorphic and differentiated the hybrids from its parental lines. The high quality genomic DNA thus obtained with Mini Prep method was found to be good for PCR applications and can be used for genetic purity and hybridity testing of cotton using molecular markers. It can also be used for genotypic selection that requires rapid screening of large populations. Acknowledgments We are thankful to Drs. Akshay Talukdar and Neelu Jain, Senior Scientists, Division of Genetics, IARI for providing valuable suggestions and selfless help to us.
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An Efficient and Rapid Method of DNA Extraction Mauro MC, Strefeler M, Weeden NF and Reisch BI (1992). Genetic analysis of restriction fragment length polymorphisms in Vitis. J Hered 83:18-21. Mazur BJ and Tingey SV (1995). Genetic mapping and introgression of genes of agronomic importance. Curr. Opinion Biotech 6: 175–182. Mullis KB, Faloona F, Scharf S, Saiki R, Horn G and Erlich H (1986). Specific enzymatic amplification of DNA in vitro. The polymerase chain reaction. Cold Spring Harbour Sym. Quart Biol 51: 263. Murray MG and Thompson WF (1980). Rapid isolation of high molecular weight DNA. Nucleic Acids Res 8: 4321-4325. Panaud O, Chen X and Mc Couch SR (1996). Development of microsatellite markers and characterization of single sequence length polymorphism (SSLP) in rice (Oryza sativa L.). Mol Gen Genet 252: 597–607.
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