Isolation and Quantification of Nucleic Acids in Plants

Chris Yuen, Ph.D.

June 13, 2006 Advances in Bioscience Education Leeward Community College

Replication

The Central Dogma of Genetics

non-coding RNA (rRNA, tRNA, siRNA, etc.)

Transcription

mRNA

Translation

Deoxyribonucleotide Ribonucleotide

Plants cells contain three distinct sets of DNA:

nuclear, plastidic, mitochondrial

The cell interior is separated from its surrounding environment by a phospholipid bilayer:

the plasma membrane

Phospholipids of the plasma membrane are amphipathic, containing both a polar (hydrophilic) head and a nonpolar (hydrophobic) tail.

Plant cells are enclosed within a rigid extracellular polysaccharide matrix:

the cell wall

Cellulose microfibrils, the main constituent of plant cell walls, as viewed through an electron microscope

Nucleic Acid Extraction Requirements 1. Disruption of cell wall and membranes to liberate cellular components.

2. Inactivation of DNA- and RNA-degrading enzymes (DNases, RNases).

3. Separation of nucleic acids from other cellular components.

• Extraction/Precipitation method • Adsorption Chromatography method

Getting Prepared: Creating a Nuclease-Free Environment

Living organisms produce several enzymes designed to degrade DNA and RNA molecules. There are several things you can do to minimize the risk of exposing your samples to external DNases and RNases.

• Autoclave solutions. This is usually sufficient for getting rid of DNases, and most RNases as well. • Treat solutions with 0.1% DEPC. DEPC inactivates nucleases by covalently modifying the His residues in proteins. Generally considered unnecessary for DNA extraction. Not compatible with solutions containing Tris or HEPES.

• Have a dedicated set of pipettors or use aerosol barrier tips.

• Wear gloves. You should be doing this anyway for safety reasons, but skin cells also produce RNase7, a potent RNA-degrading enzyme.

• Bake glass, metal, or ceramic equipment at high temp.

Overview of the Extraction/Precipitation Method

Extraction/Precipitation Method

Step 1: Disruption of cell walls by grinding Step 1+2: mechanical disruption and homogenization in extraction buffer

Grind sample into a fine powder to shear cell walls and membranes

Step 2: Lysis of cells in extraction buffer

Mix thoroughly with extraction buffer to dissolve cell membranes and inhibit nuclease activity A homogenizer allows cells to be mechanically disrupted within the extraction buffer Crude lysate

Extraction/Precipitation Method

Purposes of the Extraction Buffer

1. Dissolve cellular membranes 2. Inactivation of DNase and RNase 3. Assist in the removal of contaminants Detergents Chaotropic salts Metal chelators Salts Reducing agents CTAB PVP

Use of Detergents to Lyse Cells: Like Dissolves Like Plasma membrane (phospholipid bilayer) Detergent molecules

+

Mixed micelle

SDS

Extraction/Precipitation Method

Step 3: Organic extraction Mix thoroughly with an equal volume of organic solvent

e.g.

phenol, chloroform, or phenol:chloroform Centrifuge Aqueous Collect aqueous phase Interphase Organic Perform additional extractions for increased purity Crude lysate containing nucleic acids and other cell constituents The

aqueous phase

contains water soluble molecules, including nucleic acids. Proteins and lipids become trapped in the

organic phase

, and are thus separated away. Insoluble plant debris become trapped in the

interphase

between the two layers

Extraction/Precipitation Method

Step 4: Nucleic Acid Precipitation

Before

Supernatant Centrifuge 70% EtOH Wash Pellet Add alcohol and salt to precipitate nucleic acids from the aqueous fraction Centrifuge • Pellet down nucleic acids. • Wash pellet with 70% ethanol to remove • Discard ethanol and allow pellet to dry.

Dissolve pellet (H 2 O, TE, etc.)

Overview of the Adsorption Chromatography Method

Adsorption: the binding of molecules or particles to a surface

Basic Principle

Nucleic acids within a crude lysate are bound to a silica surface The silica surface is washed with a solution that keeps nucleic acids bound, but removes all other substances The silica surface is washed with a solution unfavorable to nucleic acid binding. The solution, containing purified DNA and/or RNA, is recovered.

Adsorption Chromatography Method

Step 1: Prepare crude lysate Step 2: Adsorb to silica surface Apply to column Centrifuge Nucleic acids Silica-gel membrane Extraction Buffer composition favors DNA and RNA adsorption to silica: • Low pH • High ionic strength • Chaotropic salt Flow through (discard) Nucleic acids bind to the membrane, while contaminants pass through the column.

Surface silanol groups are weakly acidic, and will repel nucleic acids at near neutral or high pH due to their negative charge

Adsorption Chromatography Method

Step 3: Wash away residual contaminants Centrifuge Wash buffer Nucleic acids Nucleic acids Flow through (discard) Step 4: Elute nucleic acids Elution buffer Nucleic acids Elution Buffer composition is unfavorable to surface binding:

High pH Low ionic strength

Centrifuge Nucleic acids

Using Nucleases to Remove Unwanted DNA or RNA

Add DNase + DNase (protein) Add RNase + RNase (protein) Depending on when nuclease treatment is performed, it may be necessary to repeat purification steps for protein removal (e.g. phenol/chloroform extraction).

Assessing the Quality and Yield of Nucleic Acids

Nucleic Acid Analysis via UV Spectrophotometry

DNA Absorption Spectra

By measuring the amount of light absorbed by your sample at specific wavelengths, it is possible to estimate the concentration of DNA and RNA. Nucleic acids have an absorption peak at ~260nm.

[dsDNA] ≈ A 260 [ssDNA] ≈ A 260 [ssRNA] ≈ A 260 x (50 µg/mL) x (33 µg/mL) x (40 µg/mL)

How pure is your sample?

The A 260 /A 280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. Ratios lower than 1.7 usually indicate significant protein contamination.

The A 260 /A 230 ratio of DNA and RNA should be roughly equal to its A 260 /A 280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates).

Turbidity can lead to erroneous readings due to light interference. Nucleic acids do not absorb light at the 320 nm wavelength. Thus, one can correct for the effects of turbidity by subtracting the A 320 and A 280 .

from readings at A 230 , A 260

Checking for Degradation: DNA

genomic DNA RNA (degraded) Running your sample through an agarose gel is a common method for examining the extent of DNA degradation. Good quality DNA should migrate as a high molecular weight band, with little or no evidence of smearing.

Checking for Degradation: RNA

25S 18S Ribosomal RNA (rRNA) makes up more than 80% of total RNA samples. Total RNA preps should display two prominent bands after gel electrophoresis. These correspond to the 25S and 18S rRNAs, which are 3.4 kb and 1.9 kb in Arabidopsis (respectively). Good quality RNA will have: No evidence of smearing 25S/18S ratio between 1.8 - 2.3

Today’s Lab Objectives: Use the RNeasy Extraction Kit to isolate total RNA from

Arabidopsis thaliana

.

Determine RNA yield