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Lecture 1.

Nucleic acid extraction (DNA and RNA)


By K.H.Timotius Krida Wacana Christian University (UKRIDA) Jakarta Indonesia

Definition
Nucleic acid extraction

is the isolation and purification of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid)

Outline
1. DNA Collection, sample, and storage

2. Extraction
3. Assessment of quality and quantity 4. Nucleic acid storage 5. Electrophoresis

1. DNA collection, sample and storage


Whole blood Bone marrow Serum/plasma Buccal cells Cultured cells Blood spots
Body fluids Bronchial lavage Amniotic Semen Urine
Tissue samples Fresh/frozen Paraffin-embedded Hair (shaft/root)

1. DNA collection, sample and storage (cont.)


Sample Two types of tissue: fresh and preserved. The damaging action of tissue endonucleases. Endonuclease: DNase and Rnase

1. DNA collection, sample and storage (cont.)


Temperature storage for DNA Purified DNA may be refrigerated at 4oC for up to 3 years Samples kept over 3 years should be frozen at -70oC

Specimen Storage RequirementsDNA Blood, Bone Marrow, Other Fluids


2225 C Not recommended (<24 hours)

28 C

20 C NOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs). Leukocyte pellet can be frozen for up to 1 year. 70 C Not recommended NOTE: Do not freeze blood or bone marrow before lysing red blood cells (RBCs). Leukocyte pellet can be frozen for >1 year.

Suitable condition for up to 72 hours Not recommended

Specimen Storage Requirements RNA Blood, Bone Marrow, Other Fluids


2225 C Not recommended within 2 hours 28 C 20 C

Not recommended within 2 hours Not recommended 24 weeks

NOTE: Do not freeze blood or bone marrow before

lysing red blood cells (RBCs).


70 C

Preferred storage condition

NOTE: Do not freeze blood or bone marrow before

lysing red blood cells (RBCs)

Blood and bone marrow


Collection tubes are EDTA or ACD 5 15 ml Sample should not be frozen for transport 4 25oC Notes: EDTA: Ethylenediaminetetraacetic acid ACD: Acid-Citric-Dextrose

Ethylenediaminetetraacetic acid

EDTA
EDTA is used extensively in the analysis of blood.

It is an anticoagulant for blood samples.


In biochemistry and molecular biology, ion

depletion is commonly used to deactivate metaldependent enzymes, either as an assay for their reactivity or to suppress damage to DNA or proteins.

ACD
Acid Citrate Dextrose Solution (sometimes

called Anticoagulant Citrate Dextrose Solution) is a solution of citric acid, sodium citrate and dextrose in water. It is mainly used as an anticoagulant to preserve blood specimens required for tissue typing, it is also used during procedures such as plasmapheresis instead of heparin. Two different solutions (Solution A and B) are defined by the United States Pharmacopeia.

Chaotropic agent
A denaturating agent is a substance which disrupts the

three dimensional structure in macromolecules such as proteins, DNA, or RNA and denatures them. A denaturating agent is a chaotropic agent, but chaotropic agents aren't necessarily denaturating agents. Chaotropic agents disrupt the intermolecular forces between water molecules, allowing proteins and other macromolecules to dissolve more easily. Chaotropic agents interfere with stabilizing intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Chaotropic reagents include: Urea 6 - 8 mol/l; Thiourea 2 mol/l; Guanidinium chloride 6 mol/l; Lithium perchlorate 4.5 mol/l

GITC (guanidium isothyocyanate) a general protein denaturant, being a chaotropic agent,

Serum
Collection tubes with no additives

100 l 1 ml
Transported at 20 25oC

Urine
Urine container should be used for

collection At least 1 ml should be collected Transported at 4 25oC

2. Extraction
Chemical treatments cause cells

and nuclei to burst The nucleic acid is inherently sticky, and can be pulled out of the mixture This is called spooling nucleic acid

Spooled NA

Extraction
Tissue isolation, membrane

disruptionand cell lysis Paraffin-embedded tissue require deparaffinsation (heating or solvents like xylene) Blood samples Organic extraction Inorganic extraction RNA extraction

Paraffin-embedded Tissue Sections


Genetic testing, infectious disease

testing, identity testing Formalin-fixed tissue is suitable. Mercury or other heavy metal fixatives are not acceptable. Tissue sections on glass slides can be used for in situ applications and microdissection techniques.

Blood samples
WBCs

RBCs
Plasma/serum

Basic Steps in Isolating DNA from Clinical Specimens


Separate WBCs from RBCs, if necessary Lyse WBCs or other nucleated cells Denature/digest proteins Separate contaminants (e.g., proteins, heme) from DNA Precipitate DNA if necessary Resuspend DNA in final buffer

Membrane disruption/ lysis


Detergent SDS: sodium dodecyl

sulfate Proteolytic agents: Proteinase K

Organic extraction
Phenol-chloroform extraction

Separation of protein into organic

phase and nucleic acid into aqueous phase. Phenol pH: 7.8 = 8.0 which prevent nucleic acid from remaining in the organic phase.

High MW Genomic DNA Isolation


Typical Procedure
1 Cell Lysis

Phenol Extraction
mix sample with equal volume of sat. phenol soln retain aqueous phase optional chloroform/isoamyl alcohol extraction(s)

0.5% SDS + proteinase K (55o several hours)


2 Phenol Extraction

gentle rocking several hours


3 Ethanol Precipitation 4 RNAse followed by proteinase K 5 Repeat phenol extrac-tion and EtOH ppt aqueous phase (nucleic acids) phenol phase (proteins)
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High MW Genomic DNA Isolation


Typical Procedure
1 Cell Lysis: 0.5% SDS + proteinase K (55o several hours) 2 Phenol Extraction: gentle rocking several hours 3 Ethanol Precipitation 4 RNAse followed by proteinase K 5 Repeat Phenol Extraction and EtOH ppt
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EtOH Precipitation
2-2.5 volumes EtOH, -20o high salt, pH 5-5.5 centrifuge or spool out

SDS
Sodium dodecyl sulfate (SDS or NaDS),

sodium laurilsulfate or sodium lauryl sulfate (SLS) is an organic compound with the formula CH3(CH2)11OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products. The salt is of an organosulfate consisting of a 12-carbon tail attached to a sulfate group, giving the material the amphiphilic properties required of a detergent. Being derived from inexpensive coconut and palm oils, it is a common component of many domestic cleaning products.

SDS

DNA purification: overview

cell growth

cell harvest and lysis

DNA concentration

DNA purification

Bacterial genomic DNA prep: cell extract


Lysis: Detergents Organic solvent Proteases (lysozyme) Heat

cell extract

Genomic DNA prep: removing proteins and RNA

chloroform

Need to mix gently! (to avoid shearing breakage of the genomic DNA) Add the enzyme RNase to degrade RNA in the aqueous layer

2 ways to concentrate the genomic DNA

70% final conc.

spooling

Ethanol precipitation

Plasmids: vehicles of recombinant DNA


Bacterial cell

genomic DNA

plasmids

Non-chromosomal DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate

Plasmid purification: alkaline lysis


Alkaline conditions denature DNA Neutralize: genomic DNA cant renature (plasmids CAN because they never fully separate)

DNA purification: phenol/chloroform extraction


1:1 phenol : chloroform or 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

Phenol extraction
1. 2. Aqueous volume (at least 200 microliters) Add 2 volumes of phenol:chloroform, mix well

3.
4. 5.

Spin in centrifuge, move aqueous phase to a new tube


Repeat steps 2 and 3 until there is no precipitate at phase interface (extract aqueous layer with 2 volumes of chloroform)

Ethanol precipitation (DNA concentration)


Ethanol depletes the hydration shell surrounding DNA Allowing cations to interact with the DNA phosphates Reducing repulsive forces between DNA strands Causing aggregation and precipitation of DNA Aqueous volume (example: 200 microliters) -- add 22 microliters sodium acetate 3M pH 5.2 -- add 1 microliter of glycogen (gives a visible pellet) -- add 2 volumes (446 microliters) 100% ethanol -- mix well, centrifuge at high speed, decant liquid -- wash pellet (70% ethanol), dry pellet, dissolve in appropriate volume (then determine DNA concentration)

DNA purification: overview

cell growth

cell harvest and lysis

DNA concentration

DNA purification

Inorganic extraction
Salt precipitation

adsorption to silica surfaces, and


anion - exchange chromatography.

Adsorption Methods
nucleic acids selectively absorb to silica or resins in the presence of certain chaotropic agents or salts applications:
plasmid preps fragments after electrophoresis PCR templates
Plasmid Miniprep Protocol 1. Solubilize bacteria in alkali solution 2. Neutralize with Na-acetate 3. Centrifuge, discard pellet 4. Mix supernatant with resin + chaotropic agent 5. Wash resin 6. Elute DNA with low salt buffer

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DNA purification: silica binding

Binding occurs in presence of high salt concentration, and is disrupted by elution with water

DNA Purification Method Comparison


Liquid Phase
(Lyse RBCs)

Solid Phase
(Pre-lyse cells)

Lyse cells
(Protein digestion-ProK)

Apply sample
Wash

Separate proteins from DNA


Precipitate DNA-alcohol

Elute DNA

Rehydrate DNA

Isolation of RNA
Special Considerations
RNAse inhibitors! extraction in guanidine salts phenol extractions at pH 5-6 (pH 8 for DNA) treatment with RNase-free DNase selective precipitation of high MW forms (rRNA, mRNA) with LiCl oligo-dT column

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RNA Isolation Methods Cesium Chloride Gradient


Used mainly to get clean RNA for Northern blots Homogenize cells in guanidinium isothiocyanate and

b-mercaptoethanol solution. Add to CsCl gradient and centrifuge for 1220 hours; RNA will be at the bottom of tube. Re-dissolve in TE/SDS buffer. Precipitate RNA with salt and ethanol, then rehydrate. Advantage: high quality Disadvantages: extremely time-consuming, hazardous materials disposal issues

Density Gradient Centrifugation


rate zonal/sucrose (size fractionation)
electrophoresis more common

isopycnic/CsCl (density)
density (g/cm3)
DNA ~1.7 g/cm3 protein ~1.3 g/cm3 RNA > DNA ssDNA > dsDNA GC content

1.74
1.72

1.70
1.68

20
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40

60

80

% GC base pairs

Centrifuge rotors
axis of rotation Swinging-bucket

At rest g

Spinning

Fixed-angle

Differential centrifugation of a tissue homogenate (I)


Decant supernatant

1000g/10 min

etc.

3000g/10 min

Density gradient centrifugation


Density Barrier Discontinuous Continuous

How does a gradient separate different particles?


Least dense

Most dense

Buoyant density banding Equilibrium density banding Isopycnic banding

Resolution of density gradients


Density Barrier I II Discontinuous Continuous

RNA extraction
RNA extraction demands extra care. Most

forms of RNA are labile. Contaminant RNase: pretreatment with DEPC (diethylpyrocarbonate) a strong RNase inhibitor. Glassware can be baked at 150oC for 4 hours Plastic materials can be soaked in 0.5 M NaOH for 10 min. Autoclave treatment for glassware and plastic materials.

The problem(s) with RNA:


RNA is chemically unstable -- spontaneous cleavage of phosphodiester backbone via intramolecular transesterification RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases) RNases are released upon cell lysis RNases are present on the skin RNases are very difficult to inactivate -- disulfide bridges conferring stability -- no requirement for divalent cations for activity

Top 10 sources of RNAse contamination (Ambion Scientific website)


1) 2) 3) 4) 5) 6) 7) 8) 9) 10) Ungloved hands Tips and tubes Water and buffers Lab surfaces Endogenous cellular RNAses RNA samples Plasmid preps RNA storage (slow action of small amounts of RNAse Chemical nucleases (Mg++, Ca++ at 80C for 5 +) Enzyme preparations

DEPC: diethylpyrocarbonate

RNA Isolation Methods Guanidinium-based Organic Isolation


Phenol/guanidinium solution disrupts cells,

solubilizes cell components, but maintains integrity of RNA. Add chloroform, mix, and centrifuge. Proteins/DNA remain at interface. RNA is removed with aqueous top layer. RNA is precipitated with alcohol and rehydrated. Advantage: faster than CsCl method Disadvantages: fume hood required, hazardous waste disposal issues

RNA Isolation Methods Nonorganic Salt Precipitation


Cell membranes are lysed and proteins are

denatured by detergent (such as SDS) in the presence of EDTA or other RNase inhibitors. Proteins/DNA are precipitated with a high concentration salt solution. RNA is precipitated with alcohol and rehydrated. Advantages:
Fast and easy, nontoxic Produces high quality RNA

3. Assessment of quality and quantity


Maximal absorption of nucleic acid is at wavelength

269 nm. Proteins absorb well at 280 nm. OD260 of 1.0 corresponds to approx 50 g/ml of double-stranded DNA or 40 g/ml for single-stranded DNA or RNA. OD 260/280 ratio provides an estimate of nucleic acid purity, with a pure preparation having a ratio between 1.8 and 2.0. Dyes that bind nucleic acid are acridine orange, daminoibenzoic acid (DABA), propidium iodide, and ethydium bromide.

Double-stranded and single-stranded DNA differ in their optical absorption at 260 nm

dA dG

dU dC

nucleotides ssDNA dsDNA

The conjugated p-electron systems of the purine & pyrimidine bases absorb strongly in the UV.

The absorbance of double-stranded DNA (dsDNA) at 260 nm is less than that of either single-stranded DNA (ssDNA) or the free bases. This is called hypochromism.

Using Spectroscopy to analyze DNA


DNA absorbs UV light with a major peak at 260 nm

Optical Density

This absorption is useful because it varies with the structure of DNA (&RNA) i.e. extinction coefficient depends on the structure

Wave Length
dsDNA Low extinction coefficient
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ssDNA Higher extinction coefficient

Resuspending Final Nucleic Acid Samples


Have some idea of expected nucleic acid yield. Choose diluent volume according to desired concentration. Calculating Expected DNA Yield Example: 1 X 107 cells X 6 pg DNA/cell X 80% yield= 48 mg DNA Resuspend DNA in TE buffer or ultra pure

DNAse-free water. Resuspend RNA in ultra pure RNase-free water.

Quantity from UV Spectrophotometry


DNA and RNA absorb maximally at

260 nm. Proteins absorb at 280 nm. Background scatter absorbs at 320 nm.

Quantity from UV Spectrophotometry


[DNA] =

(A260 A320) X dilution factor X 50 g/mL [RNA] = (A260 A320) X dilution factor X 40 g/mL Concentration = g of DNA or RNA per mL of hydrating solution

Evaluation of Nucleic Acids


spectrophotometrically
quantity quality

fluorescent dyes
gel electrophoresis

A260 DNA A260/A280 A260 RNA A260/A280


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1.0 50 g/ml 1.6 - 1.8 1.0 40 g/ml ~2.0

Quantity from UV Spectrophotometry Calculating Yield


Multiply the concentration of the DNA or RNA sample by the volume of hydrating solution added. Example for DNA: 150 g/mL X 0.1 mL = 15 g
Concentration from UV Spec. (g DNA per ml of hydrating solution) Volume of hydration solution DNA yield

Quality from UV Spectrophotometry


A260/A280 = measure of purity
(A260 A320)/(A280 A320) 1.7 2.0 = good DNA or RNA <1.7 = too much protein or other contaminant (?)

4. Nucleic acid storage


To prevent enzymatic or physical damage to the

purified product. Chelating agents, chaotrophic agents, refrigeration. DNA can be stored for long periods in a TRIS=EDTA buffer at 4oC. RNA, more labile, should be stored at -80oC in similar buffer. DNA and RNA can be stored as an ethanol precipitate, with -20oC being the optimal storage temperature.

Nucleic Acid Storage Requirements: Storage of DNA Specimens


<4 Months 13 Years
225 C 28 C

<7 Years
20 C

>7 Years
70 C

Not recommended

Recommended for long-term storage in ethanol

5. Electrophoresis
DNA can be separated based on

size and charge The phosphate groups are negatively charged

DNA is placed in a gel and

electricity is run through


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Separates DNA (or RNA or Protein) fragments on

the basis of charge and size Because DNA is an acid, it looses protons in basic buffers; thus it has a negative charge that is uniform per unit length Agarose (a polysaccharide) or other gel matrices are difficult for large DNA fragments to move through The larger the fragment, the more difficulty it has moving through gels By placing DNA in a gel, then applying a voltage across the gel, the negatively charged DNA will move toward the anode (positive electrode) Large fragments lag behind while small fragments move through the gel relatively rapidly

Agarose
Agar consists of a mixture of

agarose and agaropectin. The predominant component agarose is a linear polymer, made up of the repeating monomeric unit of agarobiose. Agarobiose is a disaccharide made up of D-galactose and 3,6-anhydro-Lgalactopyranose. Agaropectin is a heterogeneous mixture of smaller acidic molecules that gel poorly.

Electrophoresis
Negative DNA moves toward the

positive end Smaller fragments move farther and faster

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Electrophoresis

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Gel Electrophoresis sorts DNA molecules


by size
Separation technique: separates DNA by size and charge 1.Restriction enzymes cut DNA I into fragments 2. The gel Wells made at one end. Small amounts of DNA are placed in the wells 3. The electrical field gel placed in solution and an electrical filed is set up with one neg. (-) & one pos. (+) end 4. The fragments move negatively charged DNA fragments travel toward positive end. The smaller fragments move faster.

Mixture of DNA molecules of different sizes


Power source

Longer molecules Shorter molecules

Gel

What is the electrical charge of DNA?


Negative, so DNA pieces

migrate toward the positive pole Smaller fragments move faster and travel farther than larger fragments. Fragments of different sizes appear as bands on the gel

Agarose Gel Stained with ethidium bromide (EtBR) to Visualize the DNA

slots where DNA is loaded

1000 bp 700 bp 600 bp

500 bp
Screening PCR products to test for the presence of specific DNA sequences
molecular weight Dr.Saba markers Abdi correct PCR product molecular weight markers

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Gel Electrophoresis
Wells Large Direction of DNA Travel

Small

Figure 5.1b

DNA Size from Agarose Gel Electrophoresis: Compares unknown DNA to known size standards

Lambda DNA

1 kb ladder Lambda DNA cut with Hind III 12,218 bp


6,018 bp 3,054 bp 2,036 bp 1,636 bp 1,018 bp

100 bp ladder

23,130 bp 9,416 bp 6,557 bp 4,361 bp 48,500 bp (48.5 kb) 2,322 bp 2,027 bp

1,500 bp 1,000 bp 600 bp

300 bp

100 bp 517 bp

DNA Quality from Agarose Gel Electrophoresis


Human Whole Blood DNA

Lambda DNA marker Lambda DNA cut with Hind III marker

Whole blood genomic DNA

Text Art Page 91

The electrophoretic mobility of a DNA fragment is inversely proportional to the log of its size.

3 mm

70

60

50

40

30

20

10

Figure 5.2b

Agarose gel electrophoresis


Agarose (%) 0.5 0.8 1.0 Standard 700 bp-25 Kbp 500 bp-15 Kbp 800 bp-10 Kbp 250 bp-12 Kbp 400 bp-8 Kbp NuSieve NuSieve 3:1

1.2
1.5 2.0 3.0 4.0 6.0

150 bp-6 Kbp


80 bp-4 Kbp

300 bp-7 Kbp


200 bp-4 Kbp 100 bp-3 Kbp 50 bp-1 Kbp 500 bp-1 Kbp 100 bp-500 bp 10 bp-100 bp

Pulsed Field Gel Electrophoresis


agarose gel electrophoresis is a fundamental technique in molecular

biology but is generally unable to resolve fragments greater than 20 kilobases in size (whole microbial genomes are usually greater than 1000 kilobases in size) PFGE (pulsed field gel electrophoresis) is a adaptation of conventional agarose gel electrophoresis that allows extremely large DNA fragments to be resolved (up to megabase size fragments) essential technique for estimating the sizes of whole genomes/chromosomes prior to sequencing and is necessary for preparing large DNA fragments for large insert DNA cloning and analysis of subsequent clones also a commonly used and extremely powerful tool for genotyping and epidemiology studies for pathogenic microorganisms

Principle of PFGE
two factors influence DNA migration rates through conventional gels

- charge differences between DNA fragments - molecular sieve effect of DNA pores

DNA fragments normally travel through agarose pores as spherical

coils, fragments greater than 20 kb in size form extended coils and therefore are not subjected to the molecular sieve effect
the charge effect is countered by the proportionally increased

friction applied to the molecules and therefore fragments greater than 20 kb do not resolve
PFGE works by periodically altering the electric field orientation
the large extended coil DNA fragments are forced to change

orientation and size dependent separation is re-established because the time taken for the DNA to reorient is size dependent

Principle of PFGE
the most important factor in PFGE resolution is switching time,

longer switching times generally lead to increased size of DNA fragments which can be resolved switching times are optimised for the expected size of the DNA being run on the PFGE gel switch time ramping increases the region of the gel in which DNA separation is linear with respect to size a number of different apparatus have been developed in order to generate this switching in electric fields however most commonly used in modern laboratories are FIGE (Field Inversion Gel Electrophoresis) and CHEF (Contour-Clamped Homogenous Electrophoresis)

CHEF Switch Time Electric Field 1 Electric Field 2

+ + +

+
+ +

But, what if you want to separate larger fragments, such as entire yeast chromosomes? Pulsed-field gel electrophoresis (PFGE) can resolve fragments from 200 Kpb (0.2 Mbp) to 6000 Kbp (6 Mbp).

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Isolation of Nucleic Acids


Goals:
removal of proteins DNA vs RNA isolation of a specific type of DNA (or RNA)

Types of Methods:
differential solubility adsorption methods density gradient centrifugation

Types of DNA:
genomic (chromosomal) organellar (satellite) plasmid (extra-chromosomal) phage/viral (ds or ss) complementary (mRNA)

General Features:
denaturing cell lysis (SDS, alkali, boiling, chaotropic) enzyme treatments - protease - RNase (DNase-free) - DNase (RNase-free)

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Downstream Applications
After DNA is extracted, it is used as a

template in further molecular techniques such as


PCR (polymerase chain reaction) RFLP (restriction fragment length polymorphism) Southern Blotting

What do we need DNA for?


Detect, enumerate, clone genes Detect, enumerate species Detect/sequence specific DNA regions Create new DNA constructs (recombinant DNA)
DEPC: diethylpyrocarbonate

What about RNA?


Which genes are being transcribed? When/where are genes being transcribed? What is the level of transcription?