Nucleic Acid Extraction Methods

ISOLATION OF DNA
Miescher - first isolated DNA from human cells in 1869 by precipitation from density-gradient centrifugation
strategies
Meselson and Stahl - used such a method in 1958 to demonstrate semi-conservative replication of DNA

Preparing the Sample:

*Nucleic acid is routinely isolated from human, fungal, bacterial, and viral sources in the clinical laboratory
Bacteria and Fungi
Many of the early recombinant DNA experiments were performed with gram-negative bacteria. Methods used
at that time are the basis for a variety of DNA isolation systems currently used. Cell walls are not thick and can
be lysed by high pH and detergents.

Cell walls - can be broken mechanically by grinding or by vigorously mixing with glass beads
Gentler enzymatic methods - less likely to damage chromosomal DNA and thus are preferred for methods
involving larger chromosomal targets as opposed to plasmid DNA
Detergent (1% sodium dodecyl sulfate), strong base (0.2 M NaOH), EDTA, Glucose- can also break
bacterial cell walls.
Dilute sucrose, Triton X-100 detergent, Tris buffer, and EDTA - boiling using these after lysozyme
treatment releases DNA that can be immediately precipitated with alcohol.
DNA extracted by boiling or alkaline (NaOH) procedures is denatured (single-stranded) and may not be
suitable for methods, such as restriction enzyme analysis, that require double-stranded DNA.
Polymerase Chain Reaction (PCR) - Commercial reagent designed for isolation of DNA in amplification
procedures used for yeast, filamentous fungi,and gram-positive bacteria. The advantage of these types of
extraction is their speed and simplicity.

Viruses
Viral DNA is held within free viruses or integrated into the host genome along with host DNA. Some
procedures use cell-free specimens, such as plasma, for viral detection. Others may require concentration of
viroids by centrifugation or other methods.

Nucleated Cells in Suspension (Blood and Bone Marrow Aspirates)

WBCs - where nucleic acid in human blood or bone marrow comes from
Anticoagulants- added to the collected sample that will prevent clotting, which will trap the WBCs. Free WBCs
carrying nucleic acids and cell-free nucleic acids are available from plasma
Differential density-gradient centrifugation or differential lysis - manner of purifying WBCs in blood or
bone marrow specimens
Ficoll - a highly branched sucrose polymer that does not penetrate biological membranes used in differential
density-gradient centrifugation. Upon centrifugation, the mononuclear WBCs settle into a layer in the Ficoll
gradient that is below the less dense plasma components and above the polymorphonuclear cells and RBCs.
The layer containing the mononuclear WBCs is removed from the tube and washed by rounds of resuspension
and centrifugation in saline before proceeding with the nucleic acid isolation procedure.
Differential Osmotic Fragility of RBCs and WBCs - Another method used to isolate nucleated cells.
Incubation of whole blood or bone marrow in hypotonic buffer or water will result in the lysis of the RBCs before
the WBCs. The WBCs are pelleted by centrifugation, leaving the empty RBC membranes (ghosts) and
hemoglobin, respectively, in suspension and solution.
Plasma
With the high sensitivity achievable from amplification procedures, these sources of circulating nucleic acids
can be detected for purposes of diagnostic and prognostic analyses. Such a test is called a liquid biopsy.
Liquid biopsies may preclude surgical biopsies and allow serial biopsy testing. Isolation of cell-free nucleic acid
requires procedures to concentrate the target nucleic acid before isolation

Tissue Samples
Fresh or frozen tissue samples are dissociated before DNA isolation procedures can be started. Grinding the
frozen tissue in liquid nitrogen, homogenizing the tissue, or simply mincing the tissue using a scalpel disrupts
the whole-tissue samples. Fixed, embedded tissue may be deparaffi nized by soaking in xylene. After xylene
treatment, the tissue is rehydrated by soaking it in decreasing concentrations of ethanol. Alternatively, fixed
tissue may be used directly without dewaxing. DNA quality will also depend on how the tissue was handled
prior to fixation and the length of time the tissue was in fixation.

DNA Isolation Chemistries

Organic Isolation Methods

After release of DNA from the cell, further purification requires removal of contaminating proteins, lipids,
carbohydrates, and cell debris. For organic isolation, this is accomplished using a combination of high salt,
low pH, and an organic mixture of phenol and chloroform
Cetyltrimethylammonium bromide - used for the Isolation of small amounts of DNA from challenging
samples such as fungi. To avoid RNA contamination, RNase, an enzyme that degrades RNA, can be added at
this point. Alternatively, RNase may also be added to the resuspended DNA at the end of the procedure.
A biphasic emulsion forms when phenol and chloroform are added to the hydrophilic suspension of lysed cells
(lysate). At the proper pH, centrifugation will settle the hydrophobic phase on the bottom, with the hydrophilic
phase on top. Lipids and other hydrophobic components will dissolve in the lower hydrophobic phase. DNA will
dissolve in the upper aqueous phase. Amphiphilic components, which have both hydrophobic and hydrophilic
properties and cell debris, will collect as a white precipitate at the interface between the two layers. The DNA in
the upper phase is then precipitated by mixing with ethanol or isopropanol and salt (ammonium, potassium or
sodium acetate, or lithium or sodium chloride). The ethyl or isopropyl alcohol is added to the upper-phase
solution at 2:1 or 1:1 ratios, respectively, and the DNA forms a solid precipitate.
Carrier molecules - used for recovery of minimal amounts of DNA. Yeast RNA or glycogen was used to
coprecipitate low concentrations of DNA. Commercial preparations may be treated with DNase to avoid nucleic
acid contamination.
Centrifugation - manner through which DNA precipitate is collected. Excess salt is removed by rinsing the
pelleted nucleic acid in 70% ethanol, centrifuging, and discarding the supernatant, then dissolving the DNA
pellet in rehydration buffer, usually 10 mM Tris, 1 mM EDTA (TE), or water.

Inorganic Isolation Methods

Phenol - caustic reagent that cause safety concerns in the clinical laboratory. This should be avoided.
“salting out” - Inorganic DNA extraction. Makes use of low-pH and high-salt conditions to selectively
precipitate proteins, leaving the DNA in solution. DNA can then be precipitated using isopropanol, pelleted and
resuspended in TE buffer or water.

Solid-Phase Isolation
More rapid and comparably effective DNA extraction can be performed using solid matrices to bind and hold
the DNA for washing using silica-based products to effectively bind DNA in high-salt conditions.
Columns or Beads - modern system of solid-phase isolation make use of these two
Columns - most often small “spin columns” that fit inside microcentrifuge tubes. These columns are commonly
used to isolate viral and bacterial DNA from serum, plasma, or cerebrospinal fluid. They are also used routinely
for isolation of cellular DNA in genetics and oncology.
Preparation: starts with cell lysis and release of nucleic acids, similar to organic and inorganic procedures
Buffers - used to lyse bacterial, fungal, or animal cells
Application:

*The cell lysate is applied to a column in high-salt buffer, and the DNA in solution adsorbs to the solid matrix.
*Carrier RNA or DNA is applied with the DNA to enhance recovery, preventing irreversible binding of the small
amount of target DNA. Carrier RNA or DNA must be a nucleic acid of more than 200 nucleotides in length in
order to bind to the silica membrane.
*After the immobilized DNA is washed with buffer, the DNA is eluted in a specific volume of water, TE, ot
another low-salt buffer.
*The washing solutions and the eluant can be drawn through the column by gravity, vacuum, or centrifugal
force.
*DNA absorbed to magnetic beads is washed by suspension of the beads in buffer and collection of the beads
using a magnet applied to the outside of the tube while the buffer is aspirated or poured off.
DNA IQ system (Promega) - uses a magnetic resin that holds a specific amount of DNA (100 ng). When the
DNA is eluted in 100 μL, the DNA concentration is known (1 ng/ μ L) and ready for analysis.

Proteolytic Lysis of Fixed Material

Application:
*Thin sections are usually used for analysis, although sectioning is not necessary with very small samples such
as needle biopsies. Paraffin embedded specimens can be dewaxed with xylene or other agents and then
rehydrated before nucleic acid isolation, the fixed tissue can be used without dewaxing.
*Before lysis, cells may be washed by suspension and centrifugation in saline or other isotonic buffers.
Reagents used:
For simple screens - detergents, such as SDS or Triton
For use in PCR amplification - mixture of Tris buffer and proteinase K
Rapid Extraction Methods
PCR - new and faster methods of DNA isolation have been developed. The minimal sample requirements of
amplification procedures allow for the use of material previously not utilizable for analysis.
Chelex - cation-chelating resin that can be used for simple extraction of DNA from minimal samples.
A suspension of 10% Chelex resin beads is mixed with the specimen, and the cells are lysed by boiling.
After centrifugation of the suspension, DNA in the supernatant is cooled and may be further extracted with
chloroform before use in amplification procedures. This method is most commonly used in forensic
applications

Isolation of Mitochondrial DNA

There are two approaches to the isolation of mitochondrial DNA from eukaryotic cells.
*First approach is to isolate the mitochondria by centrifugation.
After cell preparations are homogenized by grinding on ice, the homogenate is centrifuged at low speed
(700 to 2,600 × g ) to pellet intact cells, nuclei, and cell debris. The mitochondria are pelleted from the
supernatant in a second high-speed centrifugation (10,000 to 16,000 × g ) and lysed with detergent. The
lysate is treated with proteinase to remove protein contaminants. Mitochondrial DNA can then be precipitated
with cold ethanol and resuspended in water or appropriate buffers for analysis.
*Second approach is to isolate total DNA as described previously. The preparation will contain
mitochondrial DNA that can be analyzed within the total DNA background by hybridization or PCR.