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 5М NaCl 10М Acetate ammonium (CH3COONH4)
Final concentration 0.2 M 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) Spermine or spermidine LiClO4 and acetone (for oligonucleotides only)
0.5% - 1% w/v and 0.4 М NaCl 1-10 мМ 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 • 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;
DNA
rRNA 18S, 23S, 28S
• Because of its compact size supercoiled DNA will move faster than relaxed or nicked circular forms; tRNA, 5S rRNA RNA degradation
Agarose gel DNA verification Genomic DNA
Degraded DNA