Plant DNA Extraction Plant DNA Extraction Workshop Workshop Dr. F. - - PowerPoint PPT Presentation

Plant DNA Extraction Plant DNA Extraction

Workshop Workshop

• Dr. F. Shokouhifar

Research center for Plant Sciences Research center for Plant Sciences Ferdowsi University of Mashhad

The first step in most molecular biology studies

• Detection:

PCR – PCR, – Hybridization,

• Manipulation:

– Cloning, – Transformation,

• Sequencing

Sequencing

• etc

DNA extraction

• Target nucleic acid

– DNA RNA DNA, RNA

• Source organism

– Plant (In our case ), Human, bacterial, etc.

• Starting material

– (tissue, leaf, seed, processed material, etc.)

• Desired results

Desired results

– (yield, purity, purification time required, etc.)

• Downstream application

– (PCR, cloning, labeling, blotting, etc.)

Source organism Source organism

• Eukaryotic cells
• Eukaryotic cells

– Chromosomal DNA Mit h d i l DNA – Mitochondrial DNA – Chloroplast DNA

• Prokaryotic cells

– Chromosomal DNA – Plasmid DNA

plasmids

The cell barriers for Plant DNA extraction

The main Plant DNA Extraction steps The main Plant DNA Extraction steps

• Extraction:

– Lysis of the cell wall, y , – Disturb the cell membranes, – Inactivation of cellular nucleases Inactivation of cellular nucleases, – Separation cellular debris,

• Purification:
• Purification:

– Remove contaminants from nucleic acids

Extraction methods: cell lysis Extraction methods: cell lysis

The lysis procedure should be a compromise of The lysis procedure should be a compromise of gently and rigorously methods

– To preserve the target nucleic acid – To disrupt the complex starting material

Common lysis procedures Common lysis procedures

• • Mechanical disruption

Mechanical disruption

– (e.g. grinding, hypotonic lysis)

• • Chemical treatment

Chemical treatment

– detergent to solubilise cell membranes – chaotropic salts, likes Guanidine Hydro chaotropic salts, likes Guanidine Hydro Chloride (GuHCl), Sodium dodecyl sulfate, denature proteins

• • Enzymatic digestion

– (e.g. proteinase K, )

Disruption of the cellular membrane p by detergents

Purification methods Purification methods

• Solvent extraction

– phenol and chloroform to remove proteins – isopropanol or ethanol to concentrate nucleic acids DNA acids DNA

• Chromatography

– Gel filtration, ion exchange, selective adsorption, Gel filtration, ion exchange, selective adsorption,

• Centrifugation

– ultracentrifugation in self‐forming CsCl gradients

• Affinity separation

– magnetic separation

S f Pl t DNA t ti th d Some of Plant DNA extraction methods

• Dellaporta protocol (Dellaporta et al. 1983),

CTAB t l (M d Th 1980)

• CTAB protocol (Murray and Thompson,1980),

Dellaporta method Dellaporta method

First developed by Dellaporta et al in 1983 First developed by Dellaporta et al. in 1983,

L l DNA – Large scale DNA – Pure DNA – Reliably DNA for hybridization methods likes RFLP, southern analysis, etc.

Dellaporta method steps Dellaporta method steps

• Grinding:

– Grinding freeze leaves in N2 by mortar and pestle,

• EXTRACTION:

– Tris buffer to set pH at 8 to stabilize DNA – EDTA to chalet Mg++ & Ca++ and inhibits nucleases – High NaCl concentration to Salting out Histone High NaCl concentration to Salting out Histone proteins – SDS as detergents to disrupt cell membrane H t t 60 ⁰C t h l th d t t i it – Heat at 60 ⁰C to help the detergent in its action

Dellaporta method Dellaporta method

• Purification:

Purification:

– Potassium acetate to precipitate polysaccharides, polysaccharides, – Centrifuge and Miracloth filtering to remove cell debris, remove cell debris, – Phenol/Chloroform to remove proteins – Cooled isopropanol and centrifuge to Cooled isopropanol and centrifuge to concentrate and precipitate DNA – TE buffer to resolve precipitated DNA TE buffer to resolve precipitated DNA

CTAB extraction and purification h method

• The CetylTrimethylAmmonium Bromide (CTAB)

y y ( ) protocol (Murray and Thompson,1980),

• Appropriate for the extraction and purification of

DNA from plants and plant derived foodstuff,

• suitable for the elimination of polysaccharides

and polyphenolic compounds, p yp p ,

• Results DNA with high purity and quality scale

CTAB extraction and purification h method

• Lysis

– homogenised sample – Ionic detergent (CTAB)

• Extraction
• Extraction

– Form CTAB‐DNA insoluble complex – In this step, polysaccharides, phenolic d t i d th ll l t compounds, proteins and other cell lysates dissolved in the aqueous solution are separated from the CTAB nucleic acid complex The DNA complex is solubilised by raising the salt – The DNA complex is solubilised by raising the salt concentration

• Precipitation

– precipitated with ethanol or isopropanol.

Some inhibitors of the PCR process Some inhibitors of the PCR process

Some DNA extraction tools

• Tissue grinders
• Bead Micro tube Homogenizer
• Automated DNA extraction system

Automated DNA extraction system

• FTA Cards

Tissue grinders Tissue grinders

BeadBug™ Microtube Homogenizer BeadBug Microtube Homogenizer

• mixing, lysis, grinding or homogenization

mixing, lysis, grinding or homogenization

• avoids cross contamination between samples
• Homogenize 1‐3 samples simultaneously

Homogenize 1 3 samples simultaneously

Bead Ruptor Homogenizer Bead Ruptor Homogenizer

Homogenized samples Homogenized samples

Automated DNA extraction system Automated DNA extraction system

FTATM Nucleic Acid Collection, Storage f and Purification

FTA Cards contain chemicals that:

– lyse cells, – denature proteins – protect nucleic acids from damages:

• nucleases
• oxidative
• UV

Advantages and benefits Advantages and benefits

• Capture nucleic acid in one easy step
• Captured nucleic acid is ready for downstream applications in less than 30

minutes

• DNA collected on FTA Cards is preserved for years at room temperature
• FTA Cards are stored at room temperature before and after sample

p p application, reducing the need for laboratory freezers

• Suitable for virtually any cell type
• Indicating FTA Cards change color upon sample application to facilitate

handling of colorless samples

a device for DNA capturing by the FTA Cards

Thanks for your attention Thanks for your attention Lets tea time Lets tea time