comparative studies of methods for isolation of dna from medicinal plants
TRANSCRIPT
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COMPARATIVE STUDIES OF METHODS FOR ISOLATION OF
DNA FROM MEDICINAL PLANTS
BySowjanya Boya
Under the esteemed guidance ofDr. RUPASREE MUKHOPADHYAY
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AIM AND OBJECTIVE
• To isolate pure DNA from ten medicinal plants using different methodologies.
• To compare the yield and purity of DNA from each method.
• To optimize a method that may be rapid and inexpensive with high quality and throughput.
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Andrographis paniculata Acalypha indica
Cocculus hirsutus Oxalis corniculata
Trichodesma indica
MEDICINAL PLANTS
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Cassia fistula Ipomoea carnea
Phyllanthus niruri Solanum nigrum
Vitex negundo
MEDICINAL PLANTS
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MATERIALS AND METHODS
CHEMICALS REQUIRED :
• Cetyltrimethylammonium bromide (CTAB) • NaCl • Tris-HCl • EDTA• Copper (II) acetate • Polyvinylpyrrolidone(PVP)• Sodium Acetate
• Chloroform• Isoamylalcohol• Ethanol• Isopropanol• Phenol• β-Mercaptoethanol• Liquid Nitrogen• RNAse• Proteinase K
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METHODOLOGY
SIX METHODS
Three Methods Without Using Liquid Nitrogen
Three Methods With Using
Liquid Nitrogen
• Doyle, J. J. and J. L. Doyle• Bokszczanin K, Prazybyla AA• Krizman M and et.al
• DNA Isolation using MEDOX - Kit• R.I.H. Ibrahim• Padmalatha K, Prasad M.N.V
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METHODOLOGIES WITHOUT Liq.N2
CTAB modified DNA extractions
METHOD - 1CTAB
( RNase )
METHOD - 2CTAB
( PVP and Activated Charcoal )
METHOD - 3CTAB
( copper (II) acetate solution )
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METHODOLOGIES WITH Liq.N2
DNA EXTRACTIONS
METHOD - 4DNA Isolation Using
MEDOX-EasyTM Ultrapure Genomic DNA SpinMinipreps
Kit from Plant
METHOD – 5( liquid nitrogen and
NaCl )
METHOD - 6( liquid nitrogen
and sodium acetate )
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RESULTS• Quantitative and Qualitative analysis.• Comaprision of each plant with different methods of DNA extraction.
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA
( µg/g leaf tissue )
1 0.177 0.213 0.83 4425
2 0.116 0.157 0.738 2900
3 0.068 0.088 0.7727 1700
4 0.144 0.203 0.709 1440
5 0.103 0.135 0.762 2575
6 0.191 0.205 0.931 4775
Table 1: Quantification of DNA isolated from Acalypha indica
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA
( µg/g leaf tissue )
1 0.138 0.173 0.797 3450
2 0.141 0.183 0.770 3525
3 0.042 0.058 0.7241 1050
4 0.030 0.087 0.344 300
5 0.124 0.160 0.775 3100
6 0.174 0.182 0.956 4350
Table 2: Quantification of DNA isolated from Andrographis paniculata
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Method of DNA
extractionA260 A280 A260/A280
Conc of DNA
( µg/g leaf tissue )
1 0.161 0.192 0.838 4025
2 0.157 0.193 0.813 3925
3 0.097 0.139 0.698 2425
4 0.081 0.150 0.54 810
5 0.129 0.168 0.767 3225
6 0.052 0.070 0.742 1300
Table 5: Quantification of DNA isolated from Trichodesma indicum
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA
( µg/g leaf tissue )
1 0.144 0.180 0.8 3600
2 0.140 0.156 0.897 3500
3 0.045 0.070 0.6428 1125
4 0.008 0.067 0.119 80
5 0.212 0.252 0.841 5300
6 0.268 0.291 0.920 6700
Table 4: Quantification of DNA isolated from Oxalis corniculata
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA
( µg/g leaf tissue )
1 0.136 0.173 0.78 3400
2 0.158 0.200 0.790 3950
3 0.051 0.076 0.6710 1275
4 0.039 0.104 0.375 390
5 0.075 0.104 0.721 1875
6 0.217 0.232 0.935 5425
Table 3: Quantification of DNA isolated from Cocculus hirsutus
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Table 8: Quantification of DNA isolated from Phyllanthus niruriTable 7: Quantification of DNA isolated from Ipomoea carnea
Table 6: Quantification of DNA isolated from Cassia fistula
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA
( µg/g leaf tissue )
1 0.139 0.173 0.803 3475
2 0.182 0.219 0.831 4550
3 0.178 0.231 0.770 4450
4 0.114 0.212 0.679 1140
5 0.172 0.195 0.882 4300
6 0.202 0.212 0.952 5050
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA
( µg/g leaf tissue )
1 0.131 0.170 0.77 3275
2 0.211 0.255 0.82 5275
3 0.048 0.062 0.774 1200
4 0.128 0.199 0.643 1280
5 0.168 0.194 0.865 4200
6 0.112 0.124 0.903 2800
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA
( µg/g leaf tissue )
1 0.114 0.156 0.730 3600
2 0.152 0.180 0.844 3800
3 0.057 0.075 0.76 1425
4 0.267 0.316 0.844 2670
5 0.102 0.122 0.836 2550
6 0.098 0.111 0.882 2450
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Table 10 : Quantification of DNA isolated from Vitex negundo
Table 9: Quantification of DNA isolated from Solanum nigrum
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA( µg/g leaf
tissue )
1 0.121 0.155 0.780 3025
2 0.136 0.171 0.795 3400
3 0.030 0.045 0.666 750
4 0.015 0.046 0.326 150
5 0.067 0.098 0.683 1675
6 0.175 0.188 0.930 4375
Method of DNA
extractionA260 A280 A260/A280
Conc of DNA( µg/g leaf
tissue )
1 0.121 0.155 0.780 2975
2 0.136 0.171 0.795 7375
3 0.030 0.045 0.666 900
4 0.015 0.046 0.326 130
5 0.067 0.098 0.683 1850
6 0.175 0.188 0.930 1775
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DISCUSSION
• Presence of secondary metabolites in plants reduce the yield and purity of DNA.
• Lipids (fats) found in cell membranes and proteins (enzymes and structural components) should also be separated.
• The highest yield of DNA was obtained with method 6 except for Trichodesma indica, Ipomea carnia, Phyllanthus niruri and Vitex negundo.
• Protein contamination.
PROTE
IN
CONTA
MINAT
ION
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CONCLUSION
• There is a need exist to use more better chemicals to isolate pure DNA which is completely free from contamination.
• It is necessary to establish inexpensive and less time consuming methodologies for DNA extraction from various plant parts.
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