Enclonar, Kimberly / MLS 3A
• Magnetic beads
DNA Isolation • Organic technique
January 28, 2021 o Phenol/chloroform method
Anna-Lee B. Bandoy, RMT, MSMT o Anion-exchange resin
• Inorganic technique
DNA o Salting out procedure (precipitation)
• Contains the biological instruction that make species
unique Automated Method
• It is made of four types of nucleotides which are • Vacuum-based
linked covalently into a polynucleotide chain (a DNA
• Magnetic bead-based
strand) with a sugar-phosphate backbone from which
the bases extend.
Manual Method
• Exist as a pair of molecules - shape as a double helix
Silica-based method
• Each DNA sequence contains instructions to make a
• Fast, easy-to-perform and economical
protein is known as a GENE
• Most used in the laboratory
• Employs a simple bind-wash-elute process
Sources of DNA
• The nucleic acids bind to the silica membrane in the
• Plasmid DNA
presence of chaotropic agents (disrupts hydrogen
o Small, extrachromosomal DNA molecules inside
bonds to destroy proteins)
the cell that is physically separated from
• Polysaccharides and proteins do not bind well to the
chromosomal DNA and can replicate
column and residual traces are removed during
independently
alcohol-based wash steps, along with the salts.
• Viral Nucleic Acids
• After binding and washing, nucleic acids are
• Genomic DNA from
selectively eluted under low-salt conditions, using
o Blood and Biological fluids
o Tissue and Cells water or Tris buffer
o Forensic samples • High salt - improves binding of DNA/RNA in the silica
membrane
o Plant and Fungi
o Food and feeds • Low salt - induces removal or elution of DNA/RNA
• Can use vacuum or centrifugation
Protocol to Extract DNA
• Cell lysis/dissolution
o To expose the DNA within the cell
• Removal of membrane lipids
o By adding a detergent or washing reagent
• Removal of contamination proteins
o Purify DNA that has been extracted by adding
protease
o Proteinase K (protease K or endopeptidase K) -
initial phase
▪ Commonly used to digest protein and
remove contamination from preparation of
nucleic acid
▪ Stable over a wide pH range of 4-12,
making it a widely used reagent
▪ It rapidly inactivates nucleases
• Recovery of DNA
o Precipitating the DNA with alcohol (ice cold
ethanol or isopropanol) Magnetic beads/Particle-based purification
• Methods also employ a bind-wash-elute process
• Use magnetic beads or particles functionalized with
silica surfaces to allow selective binding of DNA with
high concentration of salt.
• Dnase - removes DNA; Rnase - removes RNA
• Resolubilized DNA in a slightly alkaline buffer or in
ultra-pure water
• Adding a chelating agent to bind divalent cations
(Mg+ and Ca2+) to stop DNAse
• Cellular and histone proteins bound to the DNA can
be removed by adding a protease or by having
precipitated with sodium/ammonium acetate, or
extracted them with a phenol-chloroform mixture
prior to the DNA-precipitation.
DNA Isolation Techniques
Manual Method
• Silica-based
o Vacuum
o Centrifugation
Enclonar, Kimberly / MLS 3A
Advantages Delivers Delivers Delivers
Phenol-Chloroform Method high-purity high-purity ultrapure,
• Principle: Equal volumes of a phenol:chloroform nucleic acids nucleic acids transfection
mixture and an aqueous sample are mixed, forming a for use in for use in grade DNA for
biphasic mixture most most optimal results
• Takes longer than a column based system but has downstream downstream in sensitive
higher purity and advantage of high recovery of RNA applications applications applications
Fast, Fast, Available in
inexpensive inexpensive versatile formats
for all scales of
No silica- Available in purification.
slurry carry versatile
over, no formats for
alcohol all scales of
precipitation purification
Easy
automation
Automated DNA Isolation
Advantages
• Rapid, fully automated isolation of high-quality DNA,
RNA
• Reliable, reproducible results
• High flexibility in sample type and volume as well as
Anion-Exchange Resin elution volume and post-elution processes
• Resin is a macroporous silica-based resin with a high • Automatic pipetting of PCRs into generic 96-well PCR
density of diethylaminoethyl (DEAE) plates or strips or tubes
• Based on the interaction between negatively charged • Available from different companies
phosphates of the nucleic acid backbone and
positively charged DEAE groups on the surface of the Vacuum Devices
resin • Uses silica-based resin and columns which get
• The salt concentration and pH conditions of the automatically loaded
buffers used in each step control binding, wash • Flow through of solutions is enhanced by a vacuum
stringency and elution of nucleic acids device
• Uses alcohol to precipitate DNA • 96 preparations can be performed in parallel
Salting-out Method Magnetic bead-based
• Rapid, safe and inexpensive method
• Was developed to simplify deproteinization
procedure by S.A. Miller [Link]
• After cell lysis and a subsequent Proteinase K
digestion, the method involves salting out of the
cellular proteins by dehydration and precipitation
with saturated NaCl solution
Comparison of Nucleic Acid Purification Techniques
Silica-gel Magnetic- Anion-exchange
membrane particle resin
Technology technology
Separation Selective Binding to Solid-phase,
principle adsorption magnetic anion-exchange
to silica-gel silica chromatography
membranes particles
under
controlled
ionic
conditions
Procedure Binding: high Binding: Binding:
salt high salt Variable salt and
Elution: low Elution: low pH
salt salt Elution: Variable
Ready-to- Ready-to- salt and pH
use eluate use eluate Alcohol
precipitation