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DNA Isolation
Doc. Ruslan Kalendar
Visit Program to Egypt: FCRI-Institute of Field Crops Research, ARC, Egypt; 30.03 - 04.04.2014
DNA extraction
To understand the basic process of isolation of DNA from various sources: blood, tissue, bacteria etc.; To realise that different types of DNA require different methods of isolation; To realise that the method used is dependent upon the final application; To understand the basis of gel electrophoresis; To realise that there are different types of gel electrophoresis.
DNA structure
DNA isolation which method?
The isolation method of choice is dependent upon:
The source of the DNA: blood, tissue, bacterial, virus etc.; The final application: PCR, restriction, sequencing, fingerprinting, library construction etc.; The type of DNA: genomic vs plasmid; To a lesser extent the number of samples to be processed robotics/automation.
Isolation of DNA Methods of Isolating DNA
Tissue Homogenise, chemically or mechanically
Single cell suspension Cell wall rupture
Bacteria (Gram-) - lysozyme Yeast/fungi - zymolase
Cell membrane rupture
Detergents - SDS, sarcosine, triton X-100, CTAB Proteinases - Proteinase K, Pronase E Chelators - EDTA Guanidine thiocyanate/chloride, urea
Isolation of DNA Methods of Isolating DNA
• Cell extraction
Organic: phenol, CHCl3 (chloroform) high salt of guanidinium thiocyanate/chloride (GuTC, GuCl)
• Removal of cell debris
proteins, lipids, polysaccharides • Concentration of DNA
ethanol, isopropanol with salts: Na+, Li+, NH4+
DNA absorbing matrix (silica - silicon dioxide) CTAB, PEG, spermidine
• Optional steps
RNAse A removal of RNA
DNA extraction methods
Several common methods: • Organic extraction
• Advantage: Yields high quality DNA • Disadvantages: Toxic and time-consuming
• Chelex extraction
• Advantage: Very fast, Not toxic • Disadvantage: Product impure
• Not be suitable for DNA sequencing, restriction etc.
• Spin column extraction • Advantage: Fast, Yields high quality DNA, to be processed
robotics/automation • Disadvantages: Still toxic (GuTC) and technology consuming
Specific Methods of DNA Isolation
• Genomic DNA
SDS/Proteinase K Silica columns (Guanidine thiocyanate, CTAB) Alkaline method Automated methods
• Plasmid DNA
Alkaline/SDS Silica column methods
• Bacteriophage DNA
PEG/Salt precipitation method
Organic method
• Takes advantage of highly negative charge on nucleic acids
• Lyse cells with SDS/PK /(DTT)
• SDS = detergent (solubilizes cell membrane) • PK = proteinase K (degrades proteins • DTT = reducing agent; breaks disulfide bonds in strong proteins like
protamines
• Add equal volume of phenol • Protein fragments and lipids attracted to hydrophobic phenol • Nucleic acids attracted to water
Organic method
• Vortex and centrifuge • Remove aqueous layer • Add more phenol • Repeat procedure • Concentrate DNA
• Ethanol precipitation • Size exclusion column
Centrifugation separates layers into phenol (organic) and aqueous phases
DNA storage
Solvent:
• DNA, RNA and oligonucleotide are storage in 1xTE solution (1 mM EDTA,
Tris-HCl, pH 7.0-8.0);
Temperature: • Everyday use: +4°C; • Storage for long time: -20°C or -80°C;
• Under 70% ethanol as a pellet, DNA/RNA can be stored at +4°C almost
indefinitely;
DNA precipitation
Stock solution Final concentration
5М NaCl 0.2 M
10М Acetate ammonium (CH3COONH4) 2.0 - 2.5 M
10M LiCl 0.8 M
3M Acetate sodium (CH3COONa) 0.3 M
40% PEG 6000-8000 10 %
CTAB (Cetrimonium bromide) 0.5% - 1% w/v and 0.4 М NaCl
Spermine or spermidine 1-10 мМ
LiClO4 and acetone (for oligonucleotides only) 10 volumes of acetone with 2% LiClO4
DNA precipitation The precipitation of DNA and RNA in the presence of salt: using 2 - 3 volumes of 96% ethanol (60% - 80% final concentration of ethanol), or ½ - 2 volumes isopropanol (35% - 65% final concentration of isopropanol); Precipitation of DNA by polyethylene glycol (PEG 6000-8000) is carried out in the presence of 0.4 - 0.6 M NaCl or presence of 10 mM MgCl2, final concentrations of PEG 6000 from 5% to 15% solution; The mixture was stirred vigorously and incubated at -20°C for several hours or overnight . DNA or RNA is precipitated by centrifugation and the pellet wash with 70 % ethanol to remove any residual salt or PEG. The final concentration of 5M LiCl in solution precipitates only RNA without addition of ethanol.
Measurement of nucleic acid using spectrophotometry
• The bases in nucleic acids have max. absorption at 260 nm;
• Proteins have a max. absorption at 280 nm;
• Polyphenols/Polysaccharides have a max. absorption at 230 nm;
• A solution which has 50 g/ml of dsDNA has an absorption of 1 at 260 nm;
• A solution which has 40 g/ml of ssDNA has an absorption of 1 at 260 nm;
• OD260/OD280 = 1.8 (protein-free DNA);
• >1.8 - probably contaminated with RNA;
• OD260/OD230 > 2.0 (polysaccharide compounds - free DNA );
• <1.8 - contaminated polysaccharide, poor quality DNA;
Maximum wavelength absorbance for DNA
Separation of nucleic acids
• DNA molecules of different sizes can be separated by gel electrophoresis.
• Larger molecules migrate more slowly than smaller ones through the matrix of the gel.
• Gel electrophoresis includes:
Agarose
Polyacrylamide
Pulse field
• Agarose is polysaccharide which is extracted from seaweed. It is used to separate DNA fragments of 300-10,000 bp at 0.5-2%;
• The agarose form a solid matrix which allows DNA to migrate through an electric field based on size;
Visualization of DNA
• The DNA can be visualized by staining with ethidim bromide (EtBr).
• EtBr is an intercalating agent which will insert itself within the bases of the DNA and will exhibit florescence under UV light.
• ssDNA and RNA will also bind EtBr but to a lesser extent.
Polyacrylamide gels
• Polyacrylamide gel:
• Have smaller pores than agarose.
• Can separate DNA fragments which range in size from 10-500 bp;
• DNA fragments which differ in size by one nucleotide can be separated from each other.
• Polyacrylamide gel electrophoresis is also used to separate protein molecules.
Agarose gel DNA verification
RNA degradation
DNA
• Agarose gel electrophoresis can be used to separate DNA fragments of different sizes;
• Different forms of a DNA molecule of the same size can also be separated by agarose gel electrophoresis;
• Because of its compact size supercoiled DNA will move faster than relaxed or nicked circular forms;
rRNA 18S, 23S, 28S
tRNA, 5S rRNA
Agarose gel DNA verification
Genomic DNA
Degraded DNA