DNA Extraction and Fingerprinting: Protocol

1 Plant Genomic DNA Extraction

 The genomic DNA from each genotype is isolated from young leaves of 20 days old seedlings grown in the field. DNA is extracted from genotypes using CTAB (Cetyl tri methyl ammonium bromide) method (Doyle and Doyle, 1987). The list of buffers and stock solutions is given as follows:

1.1) 1M Tris-Cl buffer, pH 8.0, 100 ml

12.114 g Tris base is dissolved in 80 ml autoclaved distilled water. The pH is adjusted to 8.0 with 6N HCl and the final volume is made up to 100 ml with distilled water. The buffer is autoclaved and then stored at 4°C.

1.2) 0.5M EDTA, 50 ml

9.3 g of EDTA Na₂ is dissolved in 30 ml of distilled water and pH is adjusted to 8.0 with NaOH pellets. It is stirred vigorously on a magnetic stirrer to ensure that all the solutes have been dissolved. Volume is made up to 50 ml, autoclaved and stored at 4°C.

1.3) 5M NaCl, 100 ml

29.25 g NaCl is added in 80 ml double distilled H₂O and dissolved by shaking and heating for 2-3 min. The final volume is made up to 100 ml by adding double distilled water.

1.4) DNA Extraction buffer (2X), pH 8.0, 100 ml

            5M NaCl                                 :           28 ml

            1M Tris buffer                        :           10 ml

            0.5M EDTA                            :           4 ml

            2 per cent CTAB (w/v)           :           20 ml

The pH is adjusted to 8.0 with HCl and final volume is made up to 100 ml. This solution is autoclaved for 20 min and 0.2% beta mercapto ethanol (200 ml) is added and stored at 4°C.

1.5) 70 % ethanol, 100 ml

70 ml absolute ethyl alcohol is added in 30 ml double distilled water.

1.6) 2 % CTAB

2g of CTAB is dissolved in 100 ml water, and then stirred.

1.7) Isopropanol

Stored at -20^oC in dark color bottle.

1.8) TE buffer, pH 8.0, 100 ml

1 ml of 10 mM Tris Cl buffer and 0.2 ml of 1 mM EDTA solution are added to 80 ml of double distilled water and pH is adjusted to 8.0 with 6 N HCl. Final volume is made up to 100 ml, autoclaved and stored at room temperature.

Procedure for DNA extraction

1.       2-3 grams of fresh green leaves are weighed and chopped into small pieces. They are surface sterilized first with 70% ethanol and then washed with sterile distilled water and blotted with tissue paper to remove water.

2.       The leaves are frozen in liquid nitrogen and ground into a fine powder in a pre-chilled mortar-pestle.

3.       The powder are transferred to a 30 ml polypropylene tube containing 6 ml of pre-warmed (50⁰C-55⁰C) DNA extraction buffer.

4.         The contents are mixed and tubes are incubated in a water bath at 65⁰C for 1 hour.

5.         6 ml of chloroform: isoamyl alcohol (24:1) are added in the tubes and mixed by inversion for about 15 minutes.

6.         The tubes are centrifuged at 10,000 rpm for 15 min at room temperature. The upper clear aqueous layer is carefully transferred to another fresh 30 ml autoclaved polypropylene tube.

7.         2/3rd volume of isopropanol is added and incubated overnight at -20⁰C. This is done for the precipitation of nucleic acid.

8.         After incubation, the mixture in the tube is slowly and carefully mixed which result into floating of fibrous nucleic acid. The samples are then centrifuged at 10,000 rpm for 12 minutes at room temperature to pellet the DNA.

9.         The supernatant are discarded and the pellet is washed with 70% ethanol for 20 minutes and again centrifuged at 10,000 rpm for 6 minutes at room temperature.

10.      The supernatant is drained gently and the pellet is dried by inverting the tube on a tissue paper for 10 minutes.

11.      Finally the DNA pellet is dissolved in 500 ml of high salt TE buffer and kept at -20⁰C.

2 Purification of genomic DNA

2.1) Phenol: Chloroform: Iso-amyl alcohol, 500 ml

 Tris saturated phenol                          :           25 ml

Chloroform                                         :           24 ml

Isoamyl alcohol                                  :           1 ml

Stored in a brown bottle covered with two folds of silver foil and stored at -20°C

2.2)  RNase, 1 ml

            RNase powder                                    :           10 mg

            5M Sodium acetate (pH 7.5)              :           3 µl

1M Tris (pH 7.5)                                 :           10 µl

One ml of buffer is added to the vial containing 10 mg of RNase A. Aliquots of 50 ml are made in sterile eppendorf tubes. The tubes are kept water bath at 100⁰C for 15 min to denture the contaminating DNase. The tubes are cooled slowly at room temperature and stored at -20⁰C.

Procedure     

5µl RNase (10 mg/ml) is added to the isolated DNA (400µl) and incubated at 37⁰C for 1 hour. Extra TE is added to make up the volume to approx. 700 µl in a micro-centrifuge tube to prevent the loss of DNA while purification. The DNA is purified by phenol: chloroform: isoamyl alcohol (25:24:1) treatment and precipitated with 2.5 volume of absolute ethanol (ice cold). The DNA is pelleted by centrifugation and the pellet is washed with 70% ethanol. The DNA is again dissolved in 100 ml of TE and stored at -20⁰C.

3 Qualitative analysis of genomic DNA by Agarose Gel Electrophoresis

3.1) 6X gel loading buffer, 10 ml

            Bromophenol blue (0.25 % w/v)        :           0.025 g

             Xylene cyanol (0.25% w/v)               :           0.025 g

Sucrose/ glycerol                                :           40 %

Components are dissolved in 8.0 ml of doubled distilled water. pH is adjusted to 8.0 and volume is made to 10 ml. Finally it is stored at -20⁰C.

3.2) TAE (Tris- acetate electrophoresis buffer), 50X, pH 8.0, 1l

             Tris base                                            :           242 g

            Glacial acetic acid                              :           57.1 ml

            0.5M EDTA                                        :           100 ml

Tris base, glacial acetic acid and EDTA in the concentrations given above are dissolved in double distilled water. The pH is adjusted to 8.0 with NaOH pellets. Finally double distilled water is added to make up the volume to 1000 ml.

3.3) Ethidium bromide, 1 ml

10 mg ethidium bromide is added in 1 ml double distilled water and stored at 4⁰C. Working solution for staining gel is made by adding 60 µl ethidium bromide stock (10 mg /ml) in 30 ml water.

Procedure

Agarose gel electrophoresis is done for the qualitative analysis. 0.8 % agarose gel is prepared in 1X TAE buffer. 8 μl of DNA sample is mixed with 2 μl of 6X DNA loading dye and then loaded on gel and electrophoresed in 1X TAE buffer at 80-100 volts for 3 hours and then visualized on UV- trans illuminator and photographs are taken by gel documentation system (Alpha Innotech, Alpha Imager EC).

4 Quantification of DNA

Genomic DNA is quantified using UV- spectrophotometer at 260 nm. To measure the concentration, blank is set against 2 ml of TE buffer and then O.D. (optical density, absorbance) of 10 μl DNA sample with 100 times dilution (in TE buffer) is measured at 260 as well as 280 nm. The concentration of the DNA is calculated using following equation:

Concentration of DNA (μg/ml) = OD₂₆₀ × 50 × dilution factor

 (1 OD₂₆₀ = 50 μg/ml of double stranded DNA)

The ratio of OD at 260 and 280 nm give an indication of the amount of RNA or protein contamination in the sample. A value of 1.8 is optimum for the best DNA preparation. A value of the ratio below 1.8 indicates the presence of protein in the sample while a value above 1.8 indicates that the sample has RNA contamination.

5 Primer amplification

5.1 PCR reaction

Amplifications is performed in a 25 µl reaction mixture containing 2.5 µl Taq buffer (1X) containing [10mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5mM MgCl₂], 0.8mM of dNTPs, 0.04 µM of each forward and reverse primers, 100 ng genomic DNA and 3 units/µl Taq DNA polymerase (Table 1).

Table 1. Reaction mixture for PCR amplification

Component (conc.)	Single tube (µl)
DNA template (100 ng/µl) dNTPs (10mM mix) Taq polymerase (3 units/µl) Primer forward (0.6 µM) Primer reverse (0.6 µM) Taq Buffer (1X) ddH2O	1.0 µl 2.0 µl 0.5 µl 2.0 µl 2.0 µl 2.5 µl 15.0 µl
Total	25 µl

Amplification conditions

Table 2. PCR amplification protocol for SSR primer

Cycle	Denaturation	Annealing	Polymerization
Temp.	Time	Temp.	Time	Temp	Time
First cycle	94 °C	5 min	-	-	-	-
39 cycles	94°C	1 min	55-61°C	2 min	72°C	2 min
Last cycle	-	-	-	-	72°C	10 min

5.2 Agarose gel electrophoresis of amplified products

Amplified products thus obtained are separated on 2.5% agarose gel for SSR marker using horizontal gel electrophoresis assembly. The procedure used is given below:

1.     2.5 g of electrophoresis grade agarose is weighed for SSR primer. The agarose is dissolved in 100 ml of 1 X TAE buffer in a conical flask.

2.     The agarose powder is melted by heating with continuous swirling till a clear solution  obtained.

3.     Ethidium bromide at a concentration of 5 µl/100ml mixed in the gel mixture.

4.     Molten agarose is poured on to the casting tray with a comb inserted, ensuring that no air bubbles have entrapped underneath the comb.

5.     For each well DNA sample and DNA loading dye are mixed in 8:2 ratios and loaded carefully with a micropipette.

6.     Electrophoresis is done at 80-100 volt for 3 hours in 1X TAE buffer.

7.     After 75% of the gel run, its image are viewed and its photograph is recorded in the gel documentation system.

5.3 Scoring of amplified bands

The amplified products are scored separately for each primer. The PCR products for marker analysis are scored qualitatively in each lane for presence or absence. Only clear and apparently unambiguous bands are scored for the primers. Further the molecular analysis are correlated with field screening and accordingly conclusions are drawn for each primer-genotype combination.