Isolation and Quantification of Nucleic Acids

Catherine Unabia, Ph.D.

June 12, 2007Advances in Bioscience Education

Leeward Community College

The DNA double helix

Molecule contains all of the information of life as well as to serve as a template for its own replication

Flow of informationDNAto mRNAto protein

In Eukaryotes,the mRNA transcriptis exported through nuclearpores to the cytoplasm

Nucleic Acids arekey to this essential process of life

Nucleic Acidstructure

Nucleic Acid Polymers

Purines Pyrimidines

ComplementaryBase pairs

DNA

RNA polymerase transcribes gene into mRNA

RNA

Protein

mRNArRNAtRNA

translation

Amino acids specified by code

DNA templateis used to buildcomplementaryRNA strand

Biotechnology uses nucleic acids in many ways

• Creating recombinant DNA– inserting new traits like disease resistance– repairing genetic defects

• Identifying particular genes and their functions– PDI– Which genes are involved in development of cancer?

• Mapping genes on chromosomes• Comparing gene sequences between organisms

Plants cells contain three distinct sets of DNA: nuclear, plastidic, mitochondrial

Many types of RNA are also present in the cell

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

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

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

Nucleic Acid Extraction Objectives

1. Disruption of cell wall and membranes to liberate cellular components.- Grinding or digestion of cellulose with enzyme

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

Special Problems with some Plant Tissues: Polysaccharides may be abundant and may be separated together with the nucleic acids

Pigments and Secondary Products such as polyphenols may interfere with separation, or co-separate with nucleic acids

Cells may contain active RNA or DNases that quickly cut the material you are trying to isolate!

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.

Protocols for Isolating Nucleic Acids

Extraction/Precipitation Method

Adsorption Chromatography Method

Adsorption: binding of molecules or particles to a surfaceChromatography: separation based on binding

Step 1: Disruption of cell walls by grinding

Step 2: Lysis of cells in extraction buffer

Step 1+2: mechanical disruption and homogenization in extraction buffer

Extraction/Precipitation Method

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

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

Purposes of the Extraction Buffer1. Dissolve cellular membranes2. Inactivation of DNase and RNase3. Assist in the removal of contaminants

DetergentsChaotropic saltsMetal chelators (EDTA)SaltsReducing agentsCTAB (cetyltrimethylammonium bromide)

PVP (polyvinylpyrollidine)

Extraction/Precipitation Method

+

Plasma membrane(phospholipid bilayer) Detergent molecules

Use of Detergents to Lyse Cells: Like Dissolves Like

Mixed micelle

SDS

Crude lysate containing nucleic acids and other cell constituents

Mix thoroughly with an equal volume of organic solvent

e.g. phenol, chloroform, or phenol:chloroform

Centrifuge

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

Perform additional extractions for increased purity

Collect aqueous phase

Extraction/Precipitation MethodStep 3: Organic extraction

Organic

Aqueous

Interphase

• Pellet down nucleic acids. • Pellet down nucleic acids.

• Wash pellet with 70% ethanol to remove residual salts and other contaminants.

• Pellet down nucleic acids.

• Wash pellet with 70% ethanol to remove residual salts and other contaminants.

• Discard ethanol and allow pellet to dry.

After

Add alcohol and salt to precipitate nucleic acids from the aqueous fraction

Supernatant

Pellet

70% EtOH

Dissolve pellet (H2O, TE, etc.)

Step 4: Nucleic Acid Precipitation

Extraction/Precipitation Method

Before After

Centrifuge Wash Centrifuge

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

Silica-gel membrane

Apply to column

Step 2: Adsorb to silica surface

Adsorption Chromatography Method

Centrifuge

Flow through(discard)

Nucleic acids

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

Extraction Buffer composition favors DNA and RNA adsorption to silica: • Low pH• High ionic strength• Chaotropic salt Nucleic acids bind to the

membrane, while contaminants pass through the column.

Chaotropic salts disruptmolecular structure basedon hydrogen bonds; hydrophilic interactions

Centrifuge

Nucleic acids

Step 3: Wash away residual contaminants

Adsorption Chromatography Method

Wash buffer

Nucleic acids

Flow through(discard)

Nucleic acids

Elution buffer

Elution Buffer composition is unfavorable to surface binding: High pHLow ionic strength

Step 4: Elute nucleic acids

Centrifuge

Nucleic acids

Using Nucleases to Remove Unwanted DNA or RNA

Add DNase

Add RNase

+ DNase (protein)

+ 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

Degraded DNA may be unsuitable for most uses

Nucleic Acid Analysis via UV Spectrophotometry

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] ≈ A260 x (50 µg/mL)[ssDNA] ≈ A260 x (33 µg/mL)[ssRNA] ≈ A260 x (40 µg/mL)

DNA Absorption Spectra

How pure is your sample?

The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. Ratios lower than 1.7 usually indicate significant protein contamination.

The A260/A230 ratio of DNA and RNA should be roughly equal to its A260/A280 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 A320 from readings at A230, A260 and A280.

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.

genomicDNA

RNA(degraded)

Checking for Degradation: DNA

How can we use isolated DNA?

• Restriction Digest

• Hybridization with specific probes using Southern Blot

• PCR amplification of particular genes

• Recombination, cloning

• Sequencing