Purification of DNA: Dr. James Nyabuga Nyariki

DNA purification is the process of removing impurities from isolated DNA. It is important to purify DNA samples to extract sufficient quantities for research and extend their shelf life. Common purification methods include ethanol precipitation, mini-column purification using silica, organic extraction with phenol/chloroform followed by enzymatic digestion to remove proteins, and ion-exchange chromatography to separate molecules based on charge.

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Purification of DNA: Dr. James Nyabuga Nyariki

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Clement Kipyegon

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Purification of DNA

DR. JAMES NYABUGA NYARIKI

Introduction
• DNA Purification is the process of removing impurities from isolated DNA
material
• Why DNA purification is important
• DNA purification helps extract genomic and/or plasmid DNA in the sample
quantities that your research requires.

• Purifying your DNA samples from contaminants also extends their shelf-life and
reduces the probability of error when it comes to research results

• Once cell lysis has occurred, the next step is the removal of contaminants such
as carbohydrates and other macromolecules that are present in the cell.

• The removal process is often carried out through centrifugation and

subsequent pipetting.
Methods of purification
• Ethanol precipitation: this method relies on the principle that DNA is insoluble
in alcohol, and so will precipitate out during centrifugation.
• Mini-column Purification: this is the most commonly used method, relying on
DNA’s ability to bind to solid phase material (such as silica) given the right
conditions (pH and salt level in the buffer) and follows a similar procedure as
described above.
• First, a lysis procedure breaks up cellular structures.
• Then, a buffer solution is added to the spin column to set the conditions to
allow DNA to bind to solid phase material (such as silica).
• Centrifugation forces the DNA to bind to the material, and the solution to pass
through.
• Then, water is added to remove DNA from the solid phase material.
Methods of purification
• Organic extraction and enzymatic digestion for the removal of contaminants
• It involves the addition of a mixture of phenol and chloroform (1:1) to the cell lysate
for protein separation.
• The proteins aggregate as a white mass in between the aqueous phase containing
DNA and RNA, and the organic layer.
• Given that phenol is immiscible with water, two phases (one aqueous and one
phenol) form.
• During mixing, phenol will force proteins out of the aqueous layer, thus separating
DNA from contaminating protein material.
• The mixture is separated via centrifugation, and the DNA can now be pipetted from
the aqueous layer.
• Treatment of lysate with pronase or protease, in addition to phenol/chloroform,
ensures complete removal of proteins from the extract.
• The RNA can be effectively removed by using Ribonuclease, an enzyme which rapidly
degrades RNA into its ribonucleotide subunits.
• Repeated phenol extraction is not desirable, as it damages the DNA.
Methods of purification
• Using ion-exchange chromatography
• This involves the separation of ions and polar molecules (proteins, small
nucleotides and amino acids) based on their charge.
• DNA carrying negative charge binds to the cationic resin or matrix which can be
eluted from the column by salt gradient. Gradual increase in salt concentration
detaches molecules from the resin one after another.