Extraction of genomic DNA استخلاص الدنا الجينومي

Extraction of genomic DNA

Genomic DNA was extracted from young beet seedlings using DNeasy^® Plant mini Kit for DNA isolation from plant tissue, such kit provides a fast and simple way to extract DNA from plant tissue. The simple DNeasy spin column procedure yields pure total DNA for reliable PCR in very short time and also ensures complete removal of all inhibitors of PCR and other enzymatic reactions.

Genomic DNA was extracted according to the manufacturer’s protocol with minor modifications. The DNA extraction steps were as follows:

1- Grind sample tissue to a fine powder under liquid nitrogen using a mortar and pestle. Transfer 200 mg of tissue powder to 1.5 ml eppendorf tube and allow liquid nitrogen to evaporate. Do not allow the sample to thaw.

2- Add 500 ml of AP-1 buffer (lysis buffer) and 5 ml of RNase a stock solution (100 mg/ml) which digest the RNA in the sample, no tissue clumps should be visible so vortex tube vigorously to mix the mixture and to remove any clumps.            

3- Incubate the mixture for 45 min at 65^oC in a Thermo-mixer or water bath, mix tubes 4-6 times during incubation by inverting tubes (mix by invertion). This step lyses the cells.

4- Add 150 ml of AP-2 buffer (precipitation buffer) to the lysate, mix well and then incubate for 15 min in ice. This step precipitates detergent, proteins and polysaccharides. Centrifuge the lysate for 10 min at maximum speed (14000 rpm). This will remove the majority of precipitates which can cause shearing of DNA.  

5- Apply the supernatant directly to the QIAshredder spin column (Lilac membrane) sitting in a 2 ml collection tube and centrifuge for 2 min at maximum speed (14000 rpm). QIAshredder column removes most precipitates and cell debris but a small amount will pass through and form a pellet in the collection tube.

6- Transfer flow-through fraction to a new eppendorf tube, almost 450 ml of lysate is recovered. Be careful not to disturb cell debris pellet formed in the collection tube.

7- Add 1.5 volumes of AP-3/Ethanol buffer (binding buffer) to the cleared lysate and mix by pipetting and then incubate for 10 min on ice. A precipitate may form after the addition of AP-3/Ethanol but this will not affect the DNeasy procedure. Binding buffer promoted binding of DNA to DNeasy membrane.     

8- Apply 650 ml of the previous mixture, including any precipitate which may formed onto DNeasy mini spin column (White membrane) sitting in a 2 ml collection tube and centrifuge for 1 min at 8000 rpm and then discard flow-through and reuse the collection tube. 

9- Repeat step 8 with the rest of the mixture. Discard flow-through and collection tube.

10- Place DNeasy column in a new 2 ml collection tube, add 500 ml of AW buffer (washing buffer) onto the DNeasy column as first wash and centrifuge for 1 min at 8000 rpm. Discard flow-through and reuse the collection tube.

11- Add 500 ml of AW buffer (washing buffer) to the DNeasy column as second wash and then centrifuge for 2 min at 14000 rpm to dry the column membrane. Remove DNeasy column from the collection tube carefully avoid to contact it with flow-through. Discard flow-through and collection tube. It is important to dry the membrane since residual ethanol may interfere with subsequent reactions. So it is prefer to transfer DNeasy column to a new eppendorf tube and fast spin at maximum speed to ensure that membrane is completely dry and no residual of ethanol will be carried over during elution. DNA binds to the DNeasy membrane while, contaminants such as proteins and polysaccharides are efficiently removed by two wash steps.   

12- Transfer DNeasy column to a new eppendorf tube 1.5 ml and pipette 80 ml of pre-heated (65^oC), AE buffer (elution buffer) directly onto the DNeasy column membrane. Incubate for 1 hour at 37^oC in a Thermo-mixer or water bath and then centrifuge for 1 min at 8000 rpm to elute. Incubation membrane at 37^oC for 1 hour instead of 5 min increases the final concentration of DNA as well as elution with 80 ml instead of 100 ml for one time but reduces overall yield of DNA. Pure DNA is eluted in a small volume of low salt buffer or water.         

Determination of extracted DNA

Two different methods were used to measure the concentration and purity of extracted DNA:

Determination of DNA using spectrophotometer

Working with DNA, it is required to know its concentration and purity. DNA concentration of most solutions was measured by using the conversion factor that 1.0 OD at 260 nm is equivalent to 50 mg/ml of dsDNA or 37 mg/ml of ssDNA. Pure preparations of DNA have an OD 260/OD 280 value of 1.8, if the samples are contaminated with protein or phenol, the OD 260/OD 280 will be significantly less than the 1.8 value. So, an appropriate measure of the purity of the extracted DNA was determined using Smart Spec^TM 3000 Spectrophotometer, Bio-Rad Laboratories, Hercules, California, USA. The A260/280 ratio should be fall between 1.7 -1.9.

Determination of DNA by ethidium bromide fluorescence

Ethidium bromide (EtBr), a fluorescent DNA dye which can be used for staining single or double strand of nucleic acids (DNA or RNA) by intercalating itself among nitrogenous bases and it fluorescence when exposed to UV light. DNA concentration was determined by diluting the DNA 1:5 (2 ml of DNA + 8 ml ddH2O). The DNA samples were loaded onto 0.7 % (w/v) agarose gel and run against 10 ml of a suitable DNA size marker (Lambda DNA digested with Hind III and Phi X 174 DNA digested with Hae III). This marker covers a range of DNA fragment size between 23130 bp and 310 bp as shown in Figure (2), and a range of concentration between 95 ng and 11 ng. Thus, estimation of the DNA concentration in a given sample was achieved by comparing the degree of fluorescence of the unknown DNA band with the different known bands in the DNA size marker.

Random Amplified Polymorphic DNA (RAPD)

After estimating the DNA samples concentration aliquots from each stock of DNA samples were diluted to a uniform concentration of 10 ng/μl to be used with RAPD-PCR marker . While the remnant of DNA stocks were saved by storing in a deep freezer at –20^oC.

The DNA amplification protocol was performed as described by Welsh and McClelland (1990) and Williams et al. (1990) with some modifications of Driessen et al. (2001).

Primers used in RAPD analysis

Oligonucleotide sequences of the 10-mer random primers used in this study were selected from a set of Operon kits (Operon Technologies Inc., Alameda California, USA). These primers were synthesized at Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, Egypt, on an ABI 392 DNA/RNA Synthesizer (Applied Biosystems, Foster City, California, USA). A total of twelve random 10-mer primers as indicated in Table (2) were used in the detection of polymorphism among phaseolus vulgaris genotypes.

 

 

 

 

 

Figure (2): Genomic DNA extracted from phaseolus vulgaris genotypes seedlings using DNeasy plant kit loaded onto 0.7% agarose gel. Lanes M, DNA marker (Lambda Hind III / Phi X 174 Hae III).

Preparation of PCR reactions    

Reactions were carried out in a total volume of 25 µl containing 30 ng of genomic DNA as a template, 30 pmoles of random primer, 2mM of dNTP's mix (dATP, dCTP, dTTP and dGTP, ABgene, Surrey, UK), 10 X PCR buffer, 25 mM MgCl₂, and 2 units Taq DNA polymerase (MBI Fermentas Inc., Hanover, Madison, Wisconsin, USA). A master mix was prepared in a 1.5 ml eppendorf tube according to the number of PCR reactions to be performed, with an extra reaction included to compensate

for the loss of part of the solution due to frequent pipetting. An aliquot of 22 µl master mix solution was dispensed in each PCR tube (0.2 ml eppendorf tube), containing 3 µl of the appropriate template DNA (10 ng/µl). PCR reaction components are presented in Table (3)

                Table (3).

M     1      2     3     4      5     6      7      8      9    10    11   12    13    M

 RAPD-PCR reaction components

PCR Components	Amount of one PCR reaction (1X)	Master mix for 13 PCR reaction (13X)
10X PCR buffer	2.5 ml	32.5 ml
MgCl2 (25mM)	1.5 ml	19.5 ml
dNTP’s mix (2mM)	2.5 ml	32.5 ml
Primer (10 pmoles/ml)	3 ml	39 ml
Taq (5 U/ml)	0.4 ml	5.2 ml
DNA (10ng/ml)	3 ml	---
ddH2O	12.1 ml	157.3
Total volume	25ml	286

PCR program and temperature profile

PCR amplification was performed in a GeneAmp^® PCR System 9700 (Applied Biosystems, Foster City, California, USA), programmed to fulfill 40 cycles after an initial denaturation cycle for 4 min at 94ºC, in this cycle dsDNA converted to ssDNA and Taq DNA polymerase was activated. Complete denaturation of DNA results in the efficient utilization of template in the first amplification cycle and in a good yield of PCR product.

Each cycle consisted of 3 steps, a denaturation step at 94ºC for 45 sec, an annealing step at 35ºC for 1 min, and an elongation or extension step at 72ºC for 2 min. After the last cycle the primer extension segment was extended to 7 min at 72ºC in the final extending cycle then followed by soaking at 4ºC until reaction removed from PCR machine.

Electrophoresis of PCR products

The amplification products were resolved by electrophoresis in a 1.5% agarose gel containing ethidium bromide (0.5 µg/ml) in 1X TBE buffer. 15 µl of each PCR product were mixed with 3 µl of loading buffer (tracking dye), and loaded onto the wells of the gel. The gel was run at 85 volts for about 3 hrs or until tracking dye reached to the end of the gel. 

Visualization and photograph of PCR products pattern

After electrophoresis the amplified RAPD-PCR products (amplicons) pattern were visualized with an UV transilluminator. The gels were photographed using a Polaroid camera (MP4 Land Camera) and Polaroid films type 57 (ASA3000). In addition, Gel Documentation System (Gel-Doc 2000 with Diversity Database software Ver. 2.1, Bio-Rad Laboratories, Hercules, California, USA) was used for gel documentation and gel analysis.

Extraction and purification of genomic DNA

Genomic DNA was isolated from leaf tissues using the 

CTAB method  (Rogers and Bendich, 1985).

1. two gm of leaf tissue were harvested, cut into small pieces and quick-freezed in liquid nitrogen. The leaves were ground to a fine powder using a mortar and pestle prechilled with liquid nitrogen.

2. Powdered tissues were transferred into a centrifuge tube containing 14 ml of DNA extraction buffer.

3. The tubes were incubated at 65^oC for 30 min.

4. Equal volume of chloroform-isoamyl alcohol (24:1) was added and the tubes were spun at 7000 rpm for 5 min.

5. The aqueous phase was transferred to new tubes, and an equal volume of isopropanol was added to precipitate the DNA.

6. The tubes were centrifuged at 7000 rpm for 5 min.

7. The supernatant was discarded and the DNA pellet was washed with 70% (v/v) ethanol.

8. The DNA was dissolved in 3 ml of TE buffer.

9. 1μl of RNase (10 mg/μl) was added to the DNA solution and the solution was incubated at 37°C for 30 min.

10. The DNA was reprecipitated with 7.5 ml ethanol and 1 ml of 3M sodium acetate.

11. The samples were centrifuged at 10,000 rpm for10min.

12. The DNA pellet was washed with 70% ethanol and air dried,

13. The DNA was dissolved in 200ul TE buffer.

 1. Preparation of solutions used in extraction and 

            purification of genomic DNA of maize

           1. Extraction buffer:

(1.4M NaCI, 20mM EDTA, l00mM Tris-HCl, pH 8.0, 3% (w/v) CTAB (Hexadecyltrimethyl ammonium bromide), 1% v/v 2-mercaptoethanol.

119.63g       NaCI

7.45 g          Na-EDTA

12.11g         Tris-HCl

30 g             CTAB

in 1 liter

The pH was adjusted to 8.0 with 0.1 M HC1

            2. 20% SDS solution:

20g SDS in 100ml d.H₂O

   3. Chloroform/isoamyl alcohol:

240 ml Chloroform, 10 ml isoamyl alcohol

           4. 1xTE buffer (l0mM Tris-HCl/lmM EDTA):

40ml           250mM Tris-HCl

2ml             0.5M Na-EDTA

The volume was completed to 1L with d.H₂O and the pH adjusted to 8.0 with conc. HC1

5. 3M Sodium acetate:

102.6g       Sodium acetate in 250 ml d.H₂O

pH was adjusted to 5.3 using glacial acetic acid

           2. Quantitation of DNA       

Two methods were used to measure the amount of DNA in a preparation.

   Quantitation of DNA using spectrophotometer

DNA concentration was determined using the spectrophotometer, assuming that 1.0 OD at 260 nm is equivalent to 50 μg DNA. Pure preparation of DNA has an OD OD₂₆₀/OD₂₈₀ value of 1.8, if the samples are contaminated with protein, the OD₂₆₀/OD₂₈₀ will be less than the value 1.8.

    Quantitation of DNA by ethidium bromide fluorescence

   DNA concentration was determined by diluting the DNA (l:5) (2 μl of DNA + 8ul dd. H₂O). The DNA samples were loaded in 0.7% agarose gel and run against l0 μl of a DNA size marker (Lambda DNA digested with Hind Ill and Phi X 174 DNA digested with Hae III). This marker covers a range of DNA fragment size between 23130 bp and 310 bp, and a range of concentrations between 95ng and 11ng. Thus, estimation of the DNA concentration in a given sample was achieved by comparing the degree of fluorescence of the unknown DNA band with the different bands in the DNA size marker.

. PCR analysis

The DNA extracted from putatively transformed and non-transformed control plants was subjected to the polymerase chain reaction (PCR).

  1. Primers used in PCR analysis

Two sets of primers were used to detect the HVA1 and bar genes by PCR analysis. The nucleotide sequence of the forward and reverse primers used for the detection of the two genes is presented in Table (5).Primers were synthesized at the Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, Egypt, on an ABI 392 DNA/RNA synthesizer (Applied Biosystems).

Table 5. Sequence of primers used in PCR analysis for detection of HVA1 and bar genes.

Primer	Sequence (5'-3')	Length	Expected Prod. size
HVA1a	GGA GAT CTA ACA ATG GCC TCC AAC CAG AAC CAG GGG	36	747 bp
HVA1b	GGG ATA TCT AGT GAT TCC TGG TGG TGG TG	32
Bar-1	TGC CAC CGA GGG GAC ATG CCG GC	24	484 bp
Bar-2	CCT GAA GTG GAG GCC A TG GGG	21

 2. Preparation of PCR reactions

Amplification of DNA from putatively transformed and control maize plants was performed as follows:

The reaction was carried out in a volume of 50 μl containing 50 ng of genomic DNA template, 2 uM primer, 200 uM each of dATP, dCTP, dGTP and dTTP, 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 0.2 mM MgCl₂ 0.001% gelatin and 5 units of Taq polymerase enzyme (Promega).

1- A master mixture was prepared in a 1.5 ml microcentrifuge tube, so that each reaction contained:

Component                                 Amount for PCR reaction

H₂O                                             29.5μl

10x reaction buffer                     5.0 μl

MgCl₂                                          6.0 μl

    dNTP's mix.                                 4.0 μl

Primer 1                                                1 μl

Primer 2                                                1 μl

    Taq polymerase                           1 μl

2- An aliquot of 47.5 μl master mix was dispensed in each PCR tube (0.2 ml).

3- 2.5 μl of the appropriate template (50ng) were added for each reaction tube.

 3. PCR programs and temperature profiles

Amplification was carried out in a Hybaid PCR Express thermal cycler programmed for 35 cycles with the following temperature profile: 

1. An initial strand separation cycle at 94°C for 5 min.

2. This was followed by 33 cycles including a denaturation step at 94°C for 1 min., an annealing step at (55°C for 2 min. for the bar gene and 58°C for 45 sec. for the HVA1 gene) and a polymerization step at 72°C for 2 min.

3. The final cycle was a polymerization cycle performed at 72°C for 8 min.

 4. Electrophoresis of PCR products

The PCR products were analyzed by electrophoretic separation in a 1% agarose gel. Fifteen μl of each PCR product were mixed with 3 μl loading buffer and loaded into the wells of the gel. The gels were run at 80 volt for about one hour.

  5. Visualization and photography

After electrophoresis, the HVA1 or bar gene fragments were visualized with a UV transilluminator. The gels were photographed using a Polaroid camera (MP4 Land Camera) and Polaroid films type 57(ASA3000).