NUCLEIC-ACID ISOLATION METHODS

Michael T Madziva, PhD

Nucleic Acids
DNA RNA Genomic DNA Plasmid DNA

Isolation of Genomic DNA

Prokaryotes Viruses Bacteria Eukaryotes Yeast Plants

Animals

Why Isolate Genomic DNA?

Cloning of gene of interest - gene structure exons/introns Promoter analysis - identification of cis-elements
Southern blot analysis - gene size, rearrangement, amplification, mutations PCR analysis

Isolation of Genomic DNA From Mammalian Cells

Genomic DNA surrounded by nuclear & plasma membrane Need to lyse membrane - Physical: freeze/thaw cells - Detergent: solubilise membrane Need to separate DNA from lipids, proteins, other small molecules Need to concentrate DNA

Isolation of Genomic DNA From Mammalian Cells

If high MW molecules required, be careful of DNA degradation as well as shearing

Our method (Blin & Stafford, 1976) should yield DNA of 100-150 Kb; adequate for Southern analysis and construction of genomic libraries
Formamide & dialysis (Kupeic et al. 1987) gives higher MW DNA but final concentration very low. Have quicker methods for applications that dont require high MW DNA eg Bowtell 1987 guanidine to disrupt cells, 80 kb product

Separation of DNA From Proteins and Lipids

Phenol extraction: Add 1 vol phenol to vol of aqueous solution; mix to get an emulsion Add 1/2 vol (relative to aqueous solution) of chlorofom (to improve phase separation) and mix Spin Phase separation - proteins partition to phenol (bottom phase and interphase - form a white layer in interphase, while DNA is in aqueous phase (normally the upper layer) Repeat if necessary

Separation of DNA From Proteins and Lipids

Phenol extraction (cont)

Add to aqueous phase 1 vol of chlorofom, mix and spin (to remove phenol traces) EtOH precipitate DNA to concentrate and remove traces of phenol/chlorofom

Precipitation of DNA
Salt and EtOH (ion pairs ppt, EtOH reinforces) Add 1/10 vol. (relative to aqueous) of 3M Na acetate pH 5.2 Add 2-2.5 vol EtOH or 1 vol isopropanol Spin Wash with 70% EtOH Alternatively: Add 2 vol. 7.5M ammonium acetate (NH4+ - free nucleotides in soln) 2 vol. EtOH or 1 vol. isopropanol Spin Wash with 70% EtOH

Critical Parameters
Need to minimize activity of endogenous nucleases

Freeze tissue EDTA in solubilization buffer Minimize shearing of DNA Gentle (but thorough) mixing Avoid EtOH pptn and instead remove organic solvents and salt from DNA by dialysis

Preparation of Genomic DNA From Mammalian Tissue

Freeze tissue Liquid nitrogen or dry ice; evenness?
Crush to produce digestible pieces; thawing? Solubilise with buffer containing SDS and proteinase K (to digest most of cellular proteins) Separate from proteins by successive phenol/chlorofom extractions

Recover DNA by dialysis/EtOH pptn

Quality of Genomic DNA

Size: bigger than 100Kb At 260nm 1 O.D. = 50ug/ml dsDNA

Purity : index A260/A280 high quality DNA ratio larger than 1.8 Ratio of 1.5 indicates soln of 50% DNA 50% protein

Genomic DNA Isolation - Summary

Very large molecules - gentle methods Gentle lysis of cells and solubilisation of DNA Removal of proteins, RNA and other macromolecules by either chemical or enzymatic methods

Recovery and concentration of DNA

Summary (cont)
In general 1g tissue or 109 cells can yield 2mg DNA
DNA should be at least 100kb long Very viscous solution

DNA can be stored indefinitely in the presence of EtOH at 4OC or in TE buffer at -20OC TE buffer is a pH buffer & base hydrolysis catalysed by divalent ions

Preparation of Genomic DNA From Bacteria

As with mammalian cells (SDS, proteinase K) but since have cell wall need to lyse cell wall - lysozyme Ppt cell wall debris (polysaccharides and proteins)
DNA extraction and alcohol (EtOH or isopropanol) pptn

Uncut and cut genomic DNA

Isolation of Bacterial Plasmid DNA

Alkaline lysis method: Bacteria lysed in solution containing SDS and NaOH SDS: denatures bacterial proteins and NaOH denatures chromosomal and plasmid DNA.

Add potassium acetate, causing the covalently closed plasmid to reanneal.

The chromosomal DNA and bacterial proteins and SDS ppt. Plasmid is soluble and is later pptd with EtOH RNA: remove by treatment with RNAse A

For MIDI or MAXI preps, can ppt DNA with polyethylene glycol

Purification of Plasmid DNA by CsCl/Ethidium Bromide Equilibrium Centrifugation

Large scale preparation DNA free of most contaminants Kits: column based anion-exchange or size exclusion chromatography (Mini- & Maxi-preps)

Boiling Method for Plasmid Isolation

Treat bacteria with lysozyme and heat Chromosomal DNA remains attached to the bacterial membrane and is spun down

Plasmid is recovered from supernatant by isopropanol pptn

Isolation of mRNA
Required for gene cloning and expression analysis Major difficulty in RNA isolation is that most ribonucleases (RNases) are very stable and active enzymes that require no cofactors to function Therefore, first step in RNA isolation is to lyse cells in the presence of chemicals that will denature ribonucleases Crucial that denaturant in contact with cellular contents at moment of disruption as RNA unstable as harvest begins

Isolation of mRNA
All solutions DEPC (diethylpyrocarbonate) treated Lyse in the presence of RNase inhibitors eg placental ribonuclease inhibitor (RNAsin) Lyse cells and release cytosol, pellet nuclei/membranes, then phenol extract and EtOH pptn

If have contaminating DNA can remove with RNase-free DNase I RNA suspended & stored in safe RNase-free soln

References
Blin, N. & Stafford, D.W. 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303. Bowtell, D.D.L. 1987. Rapid isolation of eukaryotic

DNA. Anal. Biochem. 164, 53. Kupeic et al. 1987. Isolation of high molecular weight DNA from eukaryotic cells by formamide treatment and dialysis. Anal. Biochem. 164, 53.

mmadziva@curie.uct.ac.za