comparative studies of methods for isolation of dna from medicinal plants
TRANSCRIPT
COMPARATIVE STUDIES OF METHODS FOR ISOLATION OF
DNA FROM MEDICINAL PLANTS
BySowjanya Boya
Under the esteemed guidance ofDr. RUPASREE MUKHOPADHYAY
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.
Andrographis paniculata Acalypha indica
Cocculus hirsutus Oxalis corniculata
Trichodesma indica
MEDICINAL PLANTS
Cassia fistula Ipomoea carnea
Phyllanthus niruri Solanum nigrum
Vitex negundo
MEDICINAL PLANTS
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
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
METHODOLOGIES WITHOUT Liq.N2
CTAB modified DNA extractions
METHOD - 1CTAB
( RNase )
METHOD - 2CTAB
( PVP and Activated Charcoal )
METHOD - 3CTAB
( copper (II) acetate solution )
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 )
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
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
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
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
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
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.
THANK YOU