By

Dr Khaled Saleh Algariri

DNA & RNA EXTRACTION

Every living thing has DNA. That means that you have something in common with

a zebra, a tree, a mushroom and a beetle!!!!

DNA stands for:D: DeoxyriboseN: NucleicA: Acid

DNA is too small to see, but under a microscope it looks like a twisted up ladder!

Introduction

DefinitionDNA Extraction is

the isolation and purification of DNA (deoxyribonucleic acid)

DNA EXTRACTION

ExamplesDNA extraction is

used to isolate…

Mitochondrial DNAGenomic DNA

DNA can be extracted from…

Cells or tissuesEnvironmental

samples

http://www.davidkfaux.org/shetlandislandsmtDNA.html

http://faculty.uca.edu/~benw/biol1400/pictures/

DNA EXTRACTION

Non-examplesDNA Extraction is

not used to…

Isolate proteins or RNA

Give information about gene expression

http://iwrwww1.fzk.de/biostruct/

DNA EXTRACTION

Nucleic Acid Preparation Application?DNAPurity and amount of DNA required (and process used)

depends on intended application. Example applications: Tissue typing for organ transplant Detection of pathogens Human identity testing Genetic research Reverse transcription polymerase chain reaction and

Ligase chain reaction(PCR, LCR) RFLP (restriction fragment length polymorphism) Hybridization methods (Southern analysis)

DNA EXTRACTION

Nucleic Acid PreparationApplication?RNA

Amplification methods , Reverse transcription polymerase chain reaction(RT-PCR)

Hybridization methods (Northern analysis)

DNA EXTRACTION

DNA Purification Challenges1. Separating DNA from other cellular components

such as proteins, lipids, RNA, etc.2. Avoiding fragmentation of the long DNA

molecules by mechanical shearing or the action of endogenous nucleases.

Effectively inactivating endogenous nucleases (DNase enzymes) and preventing them from digesting the genomic DNA is a key early step in the purification process. DNases can usually be inactivated by use of heat or chelating agents.

DNA EXTRACTION

Nucleic Acid PurificationThere are many DNA purification methods.

All must:1. Effectively disrupt cells or tissues

(usually using detergent)

2. Denature proteins and nucleoprotein complexes(a protease/denaturant)

3. Inactivate endogenous nucleases(chelating agents)

4. Purify nucleic acid target away from other nucleic acids and protein(could involve RNases, proteases, selective matrix and alcohol precipitations)

DNA EXTRACTION

Disruption of Cells/TissuesMost purification methods disrupt cells using lysis buffer containing:

Detergent to disrupt the lipid bilayer of the cell membrane

Denaturants to release chromosomal DNA and denature proteins

Additional enzymes are required for lysis of some cell types:

Gram-positive bacteria require lysozyme to disrupt the bacterial cell wall.

Yeasts require addition of lyticase to disrupt the cell wall.

Plant cells may require cellulase pre-treatment.

DNA EXTRACTION

Disruption of Cells: Membrane Disruption

Detergents are used to disrupt the lipid:lipid and lipid:protein interactions in the cell membrane, causing solubilization of the membrane.

Ionic detergents (such as sodium dodecyl sulfate; SDS) also denature proteins by binding to charged residues, leading to local changes in conformation.

DNA EXTRACTION

Protein DenaturationDenaturation = Modification of conformation to unfold protein, disrupting secondary structure but not breaking the peptide bonds between amino acid residues.

Denaturation results in:Decreased protein solubilityLoss of biological activityImproved digestion by proteasesRelease of chromosomal DNA from

nucleoprotein complexes (“unwinding” of DNA and release from associated histones)

DNA EXTRACTION

Inactivation of NucleasesChelating agents, such as EDTA, sequester

Mg2+ required for nuclease activity.Proteinase K digests and destroys all

proteins, including nucleases.

Some commercial purification systems provide a single solution for cell lysis, protein digestion/denaturation and nuclease inactivation.

DNA EXTRACTION

Removal of RNASome procedures incorporate RNase

digestion during cell lysate preparation.

In other procedures, RNase digestion is incorporated during wash steps.

DNA EXTRACTION

Basic Protocol Most DNA extraction protocols consist

of two parts1. A technique to lyse the cells gently and solubilize

the DNA2. Enzymatic or chemical methods to remove

contaminating proteins, RNA, or macromolecules

In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell.

1. Lysis2. Precipitation3. Wash4. Resuspension

DNA EXTRACTION

A comparison of DNA extraction methods used in research labs as opposed to classroom labs

Research

Lysis: grind in Liquid N2 and use detergent

Precipitation Part I: phenol/chloroform extraction to get rid of proteins

Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA

Precipitation Part III: addition of ethanol to pull DNA out of solution

Wash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.

Classroom

Lysis: grind in mortar/pestel and use detergent

Precipitation Part I: NONE (chemical are too dangerous!)

Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA

Precipitation Part III: addition of ethanol to pull DNA out of solution

Wash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.

DNA EXTRACTION

LYSIS:In DNA extraction from plants, this step commonly refers to the breaking of the cell wall and cellular membranes (most importantly, the plasma and nuclear membranes)

The cell wall (made of cellulose) is disrupted by mechanical force (for example, grinding the leaves)

Then the addition of a detergent in the which breaks down the cell membranes

Detergents are able to disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules are able to pull apart the membranes

DNA EXTRACTION

DNA purification: phenol/chloroform extraction

1:1 phenol : chloroformor

25:24:1 phenol : chloroform : isoamyl alcohol

Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer

Chloroform: increases density of organic layer

Isoamyl alcohol: prevents foaming

DNA EXTRACTION

PRECIPITATION (In a research lab): This a series of steps where DNA is separated from the rest of the cellular components

In a research lab, the first part of precipitation uses phenol/chloroform to remove the proteins from the DNA

Phenol denatures proteins and dissolves denatured proteins. Chloroform is also a protein denaturant

THIS STEP CANNOT BE PERFORMED IN CLASSROOM LABS!!

The second part of research lab DNA precipitation is the addition of salts

The salts interrupt the hydrogen bonds between the water and DNA molecules.

The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol

In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.

The DNA is pelleted by spinning with a centrifuge and the supernatant removed

PRECIPITATION (In a classroom lab): This a series of steps where DNA is separated from the rest of the cellular componentsIn a classroom lab, DNA precipitation involves the

addition of salts The salts interrupt the hydrogen bonds between the

water and DNA molecules.The DNA is then precipitated from the protein in a

subsequent step with isopropanol or ethanol In the presence of cations, ethanol induces a structural

change in DNA molecules that causes them to aggregate and precipitate out of solution.

The DNA is pelleted by spinning with a centrifuge and the supernatant removed

Note: because this protocol does not use phenol/chloroform, the DNA extracted in a classroom lab is not as “clean” as the DNA extracted in a research lab!

Washing: The precipitated DNA is laden with acetate

salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA.

Resuspension: The clean DNA is now resuspended in a buffer

to ensure stability and long term storage. The most commonly used buffer for

resuspension is called 1xTE

Washing and Resuspension:

DNA EXTRACTION

cell growthcell harvest and lysis

DNA purificationDNA concentration

DNA EXTRACTION

Bacterial genomic DNA prep: cell extractLysis:

• Detergents• Organic solvent• Proteases (lysozyme)• Heat

“cell extract”

Genomic DNA prep: removing proteins and RNA

Add the enzyme RNase to degrade RNA in the aqueous layer

Need to mix gently! (to avoid shearing breakage of the genomic DNA)

chloroform

2 ways to concentrate the genomic DNA

70% final conc.

“spooling” Ethanol precipitation

DNA EXTRACTION

Genomic DNA prep in plants -- how get rid of carbohydrates? CTAB:cetyltrimeth

ylammonium bromide, hexadecyltrimethylammonium bromide.Cationic detergent

(low ionic conditions)

N+

CH3

Br-

CH3

CH3C16H33

DNA EXTRACTION

o Successful RNA isolation depends on:1) Suppression of endogenous RNAases.2) Avoid contamination with exogenous

RNAases during extraction.

A. Samples should be processed immediately or stored at -70 degree until required.

B. Inactivation of RNAases by strong denaturing agents like urea, guanidinium hydrochloride, guanidinium isothiocyanate.

Suppression of endogenous RNAases

RNA EXTRACTION

A. Specify glassware, solutions, equipments to be used

for RNA extraction only.B. Treat water and laboratory utensils with

diethylpyrocarbonate (DEPC) which is a strong RNAase inhibitor. DEPC is a suspected carcinogen.

C. Autoclave glassware, solutions and equipments if possible.

D. Use disposable gloves, disposable plastic materials that must be RNAase free.

RNA EXTRACTIONAvoid contamination with exogenous RNAases during extraction

1) Guanidinium isothiocyanate extraction:

Cell lysis.Protein denaturation by guanidinium isothiocyanate.

Cell lysate is mixed with cesium chloride.The density of RNA in cesium chloride is much greater than of other cellular elements.During ultracentrifugation, RNA pellets at the bottom of the tube and becomes separated from other cellular components.

Methods of RNA extraction

2) RNA extraction by Trizol:

Tizol is a monophasic solution of phenol & chloroform + guanidinium isothiocyanate.The presence of phenol & chloroform will separate cell lysate into two layers:

o Upper aqueous layer containing RNA.o Organic layer containing proteins.

RNA is then precipitated from the aqueous layer by isopropyl alcohol.

3) RNA extraction by spin column:

These columns use RNA adsorbing silica or glass fiber.RNA is then eluted by elution buffer.

RNA EXTRACTION

RNA extraction by Trizol

RNA EXTRACTION

4) RNA extraction by magnetic separation technology:

Couple magnetic beads to silica.Magnetic silica beads binds RNA in the lysate.The conjugated magnetic beads are then collected by applying magnetic field.RNA is then eluted from the beads.

RNA EXTRACTION

Separate WBCs from RBCs, if necessary

Lyse WBCs or other nucleated cells in presence of protein denaturants, RNase inhibitors

Denature/digest proteins

Separate proteins, DNA, and contaminantsfrom RNA

Precipitate RNA if necessary

Resuspend RNA in final buffer

Basic Steps in Isolating RNA from Clinical Specimens

RNA EXTRACTION

RNAses►RNases are naturally occurring enzymes that

degrade RNA►Common laboratory contaminant (from

bacterial and human sources)►Also released from cellular compartments

during isolation of RNA from biological samples

►Can be difficult to inactivate

RNA EXTRACTION

RNAses►RNAses are enzymes which are small

proteins that can renature and become active.

►MUST be eliminated or inactivated BEFORE isolation.

RNA EXTRACTION

Protecting Against RNAse►Wear gloves at all times►Use RNase-free tubes and pipet tips►Use dedicated, RNase-free, chemicals►Pre-treat materials with extended heat (180 C

for several hours), wash with DEPC-treated water, NaOH or H2O2

►Supplement reactions with RNase inhibitors

RNA EXTRACTION

Total RNA►80-90% of total RNA is ribosomal RNA.►2.5-5% is messenger RNA► 15-20% is transfer RNA

RNA EXTRACTION

2–25 °C 2–8 °C –20 °C –70 °C

Recommendedfor long-term

storage in ethanol

<4 Months1–3 Years <7 Years >7 Years

Nucleic Acid Storage Requirements: Storage of DNA Specimens

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