Download A-Study-of-plant

A Study of plant DNA
Holy and sweet basil plants were grown at the National Plant Protection
Experimental Station Réduit (Ministry of Agro-Industry & Food Security). All
the necessary conditions such as nutrients and pesticides were given to the
plants for good growth and development of healthy plants.
Young, tender, unbruised and healthy leaves were picked in the morning from
the Experimental Station, kept between moist tissue paper in a plastic bag
kept away from sunlight and the fresh samples were brought to the laboratory
placed on an ice box for DNA extraction and further analysis.
Experimental analyses have been done on three different types of plant
samples,
Leaves of White Tulsi (Ocimum tenuiflorum)
Leaves of Purple Tulsi (Ocimum sanctum)
Leaves of Sweet basil (Ocimum basilicum)
(b) (c)
Figure 4: (a) white tulsi light green leaves, (b) purple tulsi: purplish leaves, (c)
Dark green leaves of sweet basil.
2.1 Morphological data collection and
charaterisation
The morphological data collection was done on field and personal
observation based on the following several phenotypic traits:
Growth habit
Plant height
Type of leaf shape and stalk
Colour of stem
Strength of stem
Color and intensity of leaves
Type of venation
Color of midrib
Length and width of leaves
2.2 Materials and Methods for DNA extraction
Several experiments were carried out which are described below however
only the optimized protocol which gives the best DNA bands results were
subjected to further analysis for PCR reaction.
2.2.1 DNA Extraction and Purification
Genomic DNA was isolated from O.tenuiflorum, o. sanctum and O.basilicum
specimems using three protocols: the modified hexadecyltrimethylammonium
bromide (CTAB) mini preparation described by Doyle and Doyle 1990, with
1% 2-mercapthoethanol as a reductant, the modified sodium dodecyl (SDS)
mini preparation method of Edwards et al. (1991) with 1% 2mercapthoetnahol as reductant and using the modified Dellaporta and Doyle
&Doyle protocol method which are described below.
The modified Dellaporta and Doyle & Doyle
protocol was used for DNA extraction.
1 ml of CTAB extraction buffer was placed in a 1.5 ml eppendorf tube and
incubated at 60 °C water bath for 10 minutes (5 replicates were done for each
leaf sample).
0.15 g of fresh young leaf tissue was ground with a pestle and mortar in liquid
forming a fine powder.
The grindate was quickly transferred into the eppendorfs containing the pre heated CTAB buffer.
2 μl of 0.2 %( v/v) b-mercaptoethanol and 2 μl of 0.2 % (v/v) PVP were added
to the mixture.
The eppendorf tubes were incubated in 60 °C water bath for 25-30 minutes
with occasional swirling every 10 minutes.
After incubation, the eppendorf tubes were removed from the water bath.
660 μl of chloroform: isoamyl alcohol (24:1) was added.
The tubes were inverted several times before centrifuged at 10,000 rpm for
10 minutes.
The supernatant was removed with a micropipette and placed into another
eppendorf tube.
Steps 7-9 were repeated.
660 μl of ice-cold isopropanol was added and the tubes were placed in –20°C
freezer for further DNA precipitation (left overnight).
The following day, the tubes were centrifuged for 30 minutes at 12,000 rpm.
The supernatant was poured off, and the DNA pellet was washed with 70%
ethanol (tubes spinned at 10,000 rpm for 5 minutes).
The alcohol was poured off and the DNA pellet was allowed to completely dry
in the centrifugal evaporator for 20 minutes.
Finally, the DNA pellet was dissolved in 50 μl sterile distilled water.
CTAB mini preparation method with 1% 2mercaptoethanol
The leaves were weighed to about 500 mg per tube into an eppendorf tube
and then dropped in liquid nitrogen for 2 min.
The weighed leaves were ground in 600 ml extraction buffer (100 mM TrisHCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 2% CTAB), and 1% 2-
mercaptoethanol) preheated to 65°C and incubated for 45 min at 65°C and
vortexed every 15 min.
The mixture was homogenized with an eppendorf homogeniser and added
about 10 - 20 mg of polyvinylpolypyrrolidone (PVPP). (Modification was made
here instead of PVPP, PVP was used)
500 ml of chloroform: isoamylalcohol (24:1) was added and mixed by
constantly swirling for 10 min and centrifuging for 5 min at 14,000 rpm.
The supernatant was removed to a clean tube.
The previous step was repeated and the supernatant was transferred again to
a clean micro tube.
The nucleic acids were precipitated by addition of 600 ml ice cold isopropanol
and centrifuging at 14,000 rpm for 5 min. The DNA pellet was washed with
1000 ml cold 70% ethanol. The pellet was then dried and later resuspended
in 100 ml sterile distilled water and put in water bath overnight at 55°C.
(c) SDS mini preparation method with 1% 2mercaptoethanol
The leaves were weighed to about 500 mg per tube into an eppendorf tube
and then dropped in liquid nitrogen for 2 min.
The weighed leaves were ground in 400 ml extraction buffer (200 mM Tris Hcl
pH 7.5, 25 mM EDTA pH 8.0, 250 mM NaCl, 10% SDS (sodium dodecyl
sulphate), and 1% 2-mercaptoethanol).
The mixture was homogenised with an eppendorf homogeniser and added
about 10-20 mg of polyvinylpolypyrrolidone (PVPP).
Another 400 ml extraction buffer was added and the homogenate vortexed
followed by centrifuging for 2 min at 14,000 rpm in order to pellet the plant
debris.
The supernatant was removed to a clean tube. An equal volume of chilled
phenol: chloroform: isoamylalcohol (25:24:1) was added.
The samples were mixed well to emulsify and then centrifuged at 10 000 rpm
for 10 min.
The nucleic acids were precipitated by addition of 600 ml ice cold isopropanol
and centrifuging at 14,000 rpm for 5 min.
The DNA pellet was washed with 1000 ml cold 70% ethanol. The pellet was
then dried and later resuspended in 100 ml sterile distilled water and put in
water bath overnight at 55°C.
2.2.2 Purification of DNA for the mini CTAB and
SDS preparation
The samples isolated using the above methods were purified as detailed. To
each tube, 500 ml chloroform: iso-amylalcohol (CIA 24:1) was added and the
contents mixed by shaking for 15 min, followed by centrifugation at 12000
rpm for 15 min. The aqueous phase was transferred to a new tube and then
200 ml 1M NaCl-TE added to the old tube and shaken for 15 min. The old
tube was centrifuged for 15 min at 12000 rpm. The aqueous phase was
transferred to the new tube and mixed, followed by centrifugation at 12000
rpm for 15 min in order to settle any remaining debris. The supernatant was
then transferred to a new tube. Ice cold isopropanol (700 ml) was added to
the sample and mixed gently, and centrifuged at 10,000 rpm for 5 min and the
supernatant discarded. Cold 75% ethanol (1000 ml) was added to the pellet
to wash it thrice, and contents centrifuged at 5000 rpm for 5 min. The ethanol
was discarded and the pellet air dried. The pellet was re-suspended in 200 ml
sterile distilled water (SDW) and incubated overnight at 55°C.
NOTE: Modifications were brought to procedure for
the extraction of DNA with the mini CTAB and SDS
protocol as well for its purifications; the overnight
incubation was not done instead after adding the
sterile distilled water the pellet was kept frozen at 20°c overnight and was then subjected for further
analysis.
2.2.2.1 RNase treatment for the mini CTAB and SDS
preparation
The DNA was treated with DNase free Ribonuclease A (10 mg/ml). Large
amounts of RNA in the sample can chelate Mg2+ and reduce the yield of the
PCR (Padmalatha and Prasad, 2006). This step removes RNA from the
isolated genomic DNA. RNase (10 μl of 10 mg/ml; Sambrook et al., 1989)
was added to 100 μl of re-suspended DNA pellet and then incubated at 37ºC
over night. Equal volume of ice-cold absolute ethanol was added to each
sample and then centrifuged at 10,000 rpm for 10 min to re-precipitate the
DNA. This was done twice. The supernatant was poured off and the DNA
pellets air-dried and re-suspended in 100 μl double sterile distilled water
(dSDW).
2.3 RNASE TREATMENT OF GENOMIC DNA
100 μl (0.1 ml) of DNA was put in an eppendorf tube (6 eppendorf tubes in
total each containing DNA sample of respective basil variety).
1 μl RNAse was added in the eppendorf containing the DNA and the mixture
was incubated at 37 °C for 1 hour.
10 μl of 3M sodium acetate was then added.
100 μl of phenol: chloroform:isoamyl (25:24:1) was added and mixed well by
inverting the eppendorf tube.
The eppendorfs were spinned at 10,000 rpm for 5 minutes.
The supernatant was collected and 100 μl of chloroform:isoamyl(24:1) was
added.
The eppendorfs were spinned again at 10,000 rpm for 5 minutes.
The supernatant was collected into another clean eppendorf and 100 μl of
cold isopropanol was added (left overnight at –20 °C for DNA precipitation)
The eppendorfs were centrifuged at maximum r pm for 30 minutes after which
the isopropanol supernatant was discarded.
The DNA pellet was washed with 70 % alcohol and the pellet was dried in the
centrifugal evaporator.
The DNA pellet was dissolved in 100 μl sterile distilled water.
Samples were stored in -20°c.
2.4 Evaluation of quality and quantity of DNA
Quality Assessment of DNA
In the experiment carried out, 10 μl DNA was mixed with 990 μl sterile
distilled H2O in a quartz cuvette for each variety and the absorbance of DNA
was read at wavelengths 230, 260 & 280 nm respectively in the
spectrophotometer. The absorbance reading of all the 3 species were taken
and the purity and quantity of isolated DNA were determined
spectrophotometrically.
Wavelength ratios showing quality of DNA
OD260/OD280 =1.8 or 1.9 Pure DNA
OD260/OD280 >1.8 DNA contaminated with RNA
OD260/OD280 <1.8 Phenol or protein
contamination
OD260/OD230 <1.8 Polysaccharides or starch
contamination
Quantity determination of DNA
The formula used to calculate the DNA concentration:
DNA concentration (μg/μl) = Optical density value at 260 nm x 0.05 x dilution
factor.
The dilution factor is 1000 divided by 10, since 10 μl DNA was diluted with
990 μl Sterile distilled water making a total volume of 1000 μl.
2.5 Electrophoresis Analyses
The DNA samples were mixed with the gel loading buffer and loaded onto a
1.5% agarose gel and left to migrate for about one hour at 90 volts. The
volumes used were 7μL of DNA and 3μL of Dye. The DNA was then stained
with Ethidium Bromide and viewed under UV light.
2.6 Preparation of an Agarose gel medium of 1.5%
concentration (See Appendix).
2.7 RAPD Marker Analysis
A set of 12 primers ( OPK-05; OPL-05; OPO-03; OPC-08; OPW-04; OPC-03;
OPC-16; OPP-20; OPA-18; OPA-10; OPB-11 and OPD-13) were used.
The DNA sample was diluted from the stock with nanopure water making up
50 μl and placed on ice.
Dilution of DNA sample = 20 ng/ μl x 50 μL + dilution with nanopure water
[DNA] 260 nm
Table 5. Optimisation Protocol for RAPD Reaction
mixture
Reagents
Stock concentration
Final Molarity
Volume per reaction tube (μl)
Volume for 3 tubes (μl)
Water -
17.7
53.1
Reaction buffer
10 x
1x
3
9
MgCl2
50 mm
3 mM
1.8
5.4
dNTP
2.5 mm
0.25 mM
3
9
Primer
10 μl
0.6 μm
1.8
5.4
DNA Taq
Polymerase
5 μ/μl
1 unit
0.2
0.6
Template DNA
20 ng/μl
50 ng
2.5
7.5
Total
30
90
The master mix was prepared on ice for a total of 3 PCR tubes as follows:
Two PCR tubes were used for each primer:
1st PCR tube: Positive control which contained 2 μl of the diluted DNA.
2nd PCR tube: Negative control which contained no DNA.
2.8 Detailed Steps of PCR (See Annex)
DNA amplification was carried programmed with 3 min at 94°C for initial
denaturation, followed by 35 cycles of 54sec at 94°C, 45 sec at 43°C, 2 min
at 72°C, and a final 5 min extension at 72°C. After amplification, the DNA
fragments were separated by electrophoresis for about 3hours under
constant voltage (90 V) in 1.5% agarose gel submersed in 1X TBE buffer. The
gels were stained with ethidium bromide solution and observed under
ultraviolet light. A 1 kb fragment size marker was used as a reference to allow
comparison among the different gels (1kb ladder).
2.9 Different molarities of Template DNA were used
for screening of primers
Table….
Reagent
Stock conc.
[ng/μl]
Final molarity
[ng]
Volume of diluted DNA per
reaction tube [μl]
Template
DNA
20
30
1.5
Template
DNA
20
40
2.0
Template
DNA
20
50
2.5
Template
DNA
20
60
3.0