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