dna isolation, purification and...

DNA isolation, purification and quantification

Angela Obiageli Eni, PhD, MRSB

Why DNA purification

Deoxyribonucleic acid (DNA) purification is an extraction process that aims to separate DNA present in the nucleus of a cell from other cellular components. DNA purification consist of three major parts

1. Gentle lysis of cells and solubilization of DNA

2. Deproteinization using enzymatic or chemical methods to remove contaminating proteins RNA and other macromolecules.

3. DNA recovery

Cell lysis

Source: Promega

EDTA is usually included in

lysis buffer to inhibit DNases

Tissue must be well dispersed and not in large lumps to permit

rapid and efficient access to enzyme or chemical

Since genomic DNA must

be of high molecular weight and of sufficient

purity

Shear force and

nuclease activity must be reduced

Polysaccharide contamination is the most

common problem that affect DNA purity and may inhibit cloning procedures

Use chloroform, CTAB

and NaCl for polysaccharide removal

For plant tissue…..

CTAB forms an insoluble complex with

nucleic acid and selectively precipitates DNA, leaving behind carbohydrates, proteins and other contaminating components. The DNA-containing

precipitate can be decomplexed by dissolving it in 1N NaCl.

• Bacterial cells grown to saturation in liquid media are

harvested by centrifugation • The collected cells are lyzed with lysozyme, EDTA, SDS

etc, • Proteins are digested using proteinase K and other

cellular components are removed using organic extraction method, or silica-based technology etc.

• The last step involves DNA precipitation to obtain pure DNA at a high concentration.

For Bacteria…..

Silica based technology

• This is a widely employed method in current kits.

• DNA adsorbs specifically to silica membrane/beads/particles in the presence of certain salts and at a particular pH.

• The cellular contaminants are removed by wash steps.

• DNA is eluted in a low salt buffer or elution buffer.

Magnetic separation • This method is based on reversibly binding DNA to a magnetic solid surface/bead/particles, which have been coated with a DNA binding antibody or a functional group that interacts specifically with DNA.

• After DNA binding, beads are separated from other contaminating cellular components, washed and finally the purified DNA is eluted using ethanol extraction.

• This method is rapid and can be automated. However, it can be more costly than other methodologies.

Storage of eluted or re-suspended DNA

DNA Quantification • DNA concentration and purity are commonly

determined using a spectrophotometer which uses the absorbance/transmission of light through a liquid to determine the concentration of a particular substance in that liquid.

• The maximal absorbance for DNA is at 260nm while protein maximally absorbs at 280nm. Therefore, the ratio of nucleic acid to protein (260/280) is generally used as an indicator of the purity of DNA samples.

• NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration and purity in as little as 1μl is most commonly used in most lab.

‘Blank’ the spectrophotometer or Nanodrop using the solution the DNA is re-suspended or eluted in. Be sure to record the concentration and purity.

• A good purity ranges from 1.80-2.00

• A 260/280 ratio of < 1.8 indicates the sample is contaminated with protein or organic solvent such as phenol.

Spectrophotometer

Tips

If two consecutive samples have significantly different concentrations, this may affected the accuracy of the NanoDrop. ‘Blank again’

DNA in a buffered solution e. g. TE buffer will give more accurate and consistent absorbance readings than DNA in water.

For a 1-cm pathlength, the optical density at 260 nm (OD260) equals 1.0 for the following solutions:

• a 50 μg/mL solution of dsDNA

• a 33 μg/mL solution of ssDNA

• a 20-30 μg/mL solution of oligonucleotide

• a 40 μg/mL solution of RNA

• DNA concentration (ug/ml) = OD260 reading x 50 x dilution factor.

A sample of dsDNA was diluted 50X. The diluted sample gave a reading of 0.65 on a spectrophotometer at OD260. To determine the concentration of DNA in the original sample, perform the following calculation:

• dsDNA concentration = 50 μg/mL × OD260 × dilution factor

• dsDNA concentration = 50 μg/mL × 0.65 × 50

• dsDNA concentration = 1.63 mg/mL

Example of concentration calculation