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