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DNA Isolation
DNA Isolation
Early years: Precipitation (Miescher)→ density gradient sucrose polymer that does not
centrifugation strategies (Meselson and Stahl)→ penetrate biological
solubility differences membranes.
o Chromosomal DNA and proteins → o Upon centrifugation→
neutralized in acetate at low pH → the mononuclear
forms large aggregates WBCs (the desired cells for isolation
o Small plasmids→ return to supercoiled of nucleic acid) settle into a layer in
state the Ficoll gradient → layer of
o Alkaline lysis – used for extensive mononuclear WBCs→ removed→
extraction of 1- 50 kb plasmid DNA washed by resuspension and
centrifugation in saline
PREPARING THE SAMPLE
- nature of the starting material determine the - Incubation of whole blood or bone marrow in
exact DNA test method. hypotonic buffer or water will result in the lysis of
the RBCs before the WBCs.
Bacteria and Fungi - WBCs: pelleted by centrifugation, leaving the
- gram negative bacteria were previously used for empty RBC membranes (ghosts) and
recombinant DNA experiments hemoglobin, respectively, in suspension and
o thin cell wall lysis → high pH and solution.
detergent
o thick cell wall lysis → enzyme products, Plasma
mechanical (grinding or by glass beads) - Solid tumors and transplanted organs release
o bacterial cell walls → broken by cells, exosomes, and nucleic acid into the
detergent (1% sodium dodecyl sulfate) bloodstream.
and strong base (0.2 M NaOH) in the - Exosomes contain proteins, lipids, and nucleic
presence of Tris base, (EDTA), and acid.
glucose o collected by centrifugation for diagnostic
- DNA extracted by boiling or alkaline (NaOH) and prognostic analyses
procedures → denatured (single stranded) → - Liquid biopsy: preclude surgical biopsies and
not for restriction enzyme analysis that require allow serial biopsy testing.
dsDNA. - Isolation of cell-free nucleic acid requires
procedures to concentrate the target nucleic acid
Viruses before isolation which include solidphase
- Location - free viruses and host genome along collection of nucleic acid on beads or columns
with host DNA.
- Specimens: cell-free specimens/plasma (for viral Tissue Samples
detection) - Whole-tissue samples : disrupted by grinding the
- Others: concentration of viroids by centrifugation frozen tissue in liquid nitrogen, homogenizing
or other methods. the tissue, or simply mincing the tissue using a
scalpel
Nucleated Cells in Suspension (Blood and Bone - Fixed, embedded tissue → deparaffinized by
Marrow Aspirates) soaking in xylene (a mixture of three isomers of
- comes mostly from WBCs dimethylbenzene) → tissue is rehydrated by
- Serum from clotted blood soaking it in decreasing concentrations of
o proteins, lipids, and other molecules, ethanol.
but not nucleic acids. - Fixed tissue → used directly without dewaxing.
- Plasma - Buffered formalin: least damaging among tested
o free WBCs carrying nucleic acids and fixatives,
cell-free nucleic acids - Mercury based fixatives (Bouin ’ s and B-5):
- differential density-gradient centrifugation or worst for DNA recovery
differential lysis - In general, DNA target products of 100 base
o ways by which WBCs in blood or bone pairs (bp) or less can consistently be obtained
marrow specimens are purified from fixed tissue.
1) Differential density-gradient - Extended digestion in proteinase K may yield
centrifugation longer fragments.
o whole blood or bone marrow mixed with
isotonic saline is overlaid with Ficoll.
DNA Isolation Chemistries o Less isopropanol is added for precipitation;
Organic Isolation Methods therefore, isopropanol can be more practical for
o Purification by using a combination of high large-volume samples.
salt, low pH, and an organic mixture of
phenol and chloroform → dissolves o Isopropanol is less volatile than ethanol and
hydrophobic contaminants, such as lipids and precipitates DNA at room temperature.
lipoproteins and collects cell debris and strips Precipitation at room temperature reduces
away most DNA-associated proteins coprecipitation of salt.
o Isolation of small amounts of DNA from fungi o The choice of which alcohol to use depends on
can be facilitated by pretreatment with the starting material, the size and amount of
cetyltrimethylammonium bromide, a cationic DNA to be isolated, and the design of the
detergent that efficiently separates DNA from method.
polysaccharide contamination.
o For low concentrations of DNA, longer
o To avoid RNA contamination, RNase, an precipitation times at freezer temperatures
enzyme that degrades RNA, can be added at may be required to maximize the amount of DNA
this point. It may also resuspended DNA at the that is recovered.
end of the procedure.
o Recovery of minimal amounts of DNA can be
o A biphasic emulsion forms when phenol and optimized using carrier molecules such as
chloroform are added to the hydrophilic yeast RNA or glycogen to coprecipitate low
suspension of lysed cells (lysate). concentrations of DNA.
o At the proper pH, centrifugation will settle the o Glycogen is a biological material derived from
hydrophobic phase on the bottom, with the mussels hence, some preparations of glycogen
hydrophilic phase on top. may contain DNA.
o DNA will dissolve in the upper aqueous phase. o Uses low-pH and high-salt conditions to
selectively precipitate proteins, leaving the DNA
o Amphiphilic components, which have both in solution.
hydrophobic and hydrophilic properties and cell
debris, will collect as a white precipitate at the o The presence of the chelating agent, EDTA,
interface between the two layers. protects the DNA from damage by DNases
present in the environment of DNA preparations
o The DNA in the upper phase → precipitated by intended for long-term storage.
mixing with ethanol or isopropanol and salt
(ammonium, potassium or sodium acetate, or o Low DNA yield→ it is better to dissolve it in
lithium or sodium chloride). water.
o The ethyl or isopropyl alcohol is added to the o Precipitation of the DNA excludes hydrophilic
upper-phase solution at 2:1 or 1:1 ratios, proteins, carbohydrates, and other residual
respectively, and the DNA forms a solid contaminants still present after protein
precipitate.
extraction.
o Ethanol is one of the general- use formulas,
o In addition, the concentration of the DNA can be
reagent grade. It is mixed with other components
controlled by adjusting the buffer or water
because pure 100% ethanol cannot be distilled.
volume used for resuspension of the pellet.
o Reagent-grade alcohol (90.25% ethanol,
o Inorganic DNA isolation does not include the
4.75% methanol, 5% isopropanol) is denatured;
organic extraction step.
o Isopropanol: undenatured, or pure, because it
o The supernatant with the DNA → mixed with
is composed of 99% isopropanol and 1% water, ethanol or isopropanol to precipitate the DNA to
with no other components. be collected and resuspended in a smaller
volume.
Solid-Phase Isolation o For solid-phase separation, the cell lysate is
o More rapid and comparably effective DNA applied to a column in high-salt buffer, and the
extraction using solid matrices to bind and hold DNA in solution adsorbs to the solid matrix.
the DNA for washing.
o Carrier RNA or DNA is applied with the DNA to
o Silica-based products: effectively bind DNA in enhance recovery, preventing irreversible
high-salt conditions. binding of the small amount of target DNA.
o Modern systems can be purchased with solid o The carrier must be a nucleic acid of more than
matrices in the form of columns or beads. 200 nucleotides in length in order to bind to the
silica membrane.
o Columns come in various sizes, depending on
the amount of DNA to be isolated. o Other carriers used to enhance precipitation,
such as glycogen and linear polyacrylamide,
o Columns used in the clinical laboratory are most cannot be used in this application.
often small “spin columns” that fit inside
microcentrifuge tubes. o After the immobilized DNA is washed with buffer,
the DNA is eluted in a specific volume of water,
o These columns are commonly used to isolate TE, or another low-salt buffer.
viral and bacterial DNA from serum, plasma,
or cerebrospinal fluid. o The washing solutions and the eluant can be
drawn through the column by gravity, vacuum,
o They are also used routinely for isolation of or centrifugal force.
cellular DNA in genetics and oncology.
o 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.
o After cleaning, glassware must be baked for four o Bacterial and fungal RNA are also isolated by
to six hours at 400°C to inactivate the RNases. chemical lysis or by grinding in liquid nitrogen.
Other RNase inhibitors can be added directly to RNA o In some isolation procedures, DNase is added at
preparations include the lysis step to eliminate contamination of DNA.
o Vanadylribonucleoside complexes- bind the Alternatively, RNF DNase also may be added
active sites of the RNase enzymes directly to the isolated RNA at the end of the
o macaloid clays- absorb the RNase proteins. procedure.
o form a stable noncovalent complex with
the RNases in solution. o After phase separation, the upper aqueous
phase containing the RNA is removed to a
RNA Isolation Chemistries clean tube, and the RNA is precipitated by
o Reticulocytes in blood and bone marrow addition of two volumes of ethanol or one
samples are lysed by osmosis or separated volume of isopropanol.
from WBCs by centrifugation.
o Glycogen or yeast-transfer RNA may be as a
o When dissociating tissue, the sample should be carrier to aid RNA pellet formation. The RNA
kept frozen in liquid nitrogen or immersed in precipitate is then washed in 70% ethanol and
buffer that will inactivate intracellular RNases. resuspended in RNF buffer or water.
This is especially true for tissues such as
Solid-Phase Isolation oligomers of thymine or uracil immobilized on a
o Begins with similar steps as described matrix resin column or beads are used
previously for organic extraction.
o The strong denaturing buffer conditions o The polyT or polyU oligomers will bind the
must be adjusted before application of the polyA tail found exclusively on mRNA. After
lysate to the column. In some procedures, washing away residual RNA, polyA RNA is
ethanol is added at this point. eluted by washing the column with warmed, low-
o Some systems provide a filter column to salt buffer containing detergent to break the
remove particulate material before application to hydrogen bonds between the mRNA and the
the adsorption column. column. With this approach, approximately 30
o As with DNA columns, commercial reagents are to 40 ng of mRNA can be obtained from 1 μ g
supplied with the columns to optimize RNA of total RNA.
adsorption and washing on the silica-based
matrix. o There are limitations to the isolation of polyA
RNA using oligo dT or dU.
o After lysate is applied to the column in the high- 1) Efficiency of polyA and polyU binding is variable.
salt chaotropic buffer, the adsorbed RNA is 2) Secondary structure (intrastrand or interstrand
washed with the supplied buffers. DNase may hydrogen bonds) in the target sample may
be added directly to the adsorbed RNA on the compete with binding to the captureligomer.
column to remove contaminating DNA. 3) mRNAs with short polyA tails may not bind
efficiently or at all. AT-rich DNA fragments might
o Washing solutions and the eluant are drawn bind to the column and not only compete with
through the column by gravity, vacuum, or the desired mRNA target but also contaminate
centrifugal force. Small columns of silica-based the final eluant.
material that fit inside microfuge tubes (spin
columns) are widelyused for routine nucleic acid MEASUREMENT OF NUCLEIC ACID QUALITY AND
isolation from all types of specimens. QUANTITY
o Consistent results require that run-to-run
o Generally, 1 million eukaryotic cells or 10 to variation be minimized. Fortunately,
50 mg of tissue will yield about 10 μ g of RNA. measurement of the quality and quantity of DNA
The yield of RNA from biological fluids will and RNA is straightforward.
depend on the concentration of
microorganisms or other target molecules Electrophoresis
present in the specimen o Fluorescent dyes such as ethidium bromide or
SybrGreen I bind specifically to DNA and are
Proteolytic Lysis of Fixed Material used to visualize the sample preparation.
o The quality of RNA from fixed, paraffin- Ethidium bromide or SybrGreen II are
embedded tissue will depend on the fixation used to detect RNA.
process and handling of the specimen before Silver stain has been used to detect
fixation. small amounts of DNA by visual
inspection.
o Commercial reagent sets are available for o The appearance of DNA on agarose gels
extraction of RNA from fixed-tissue specimens. depends on the type of DNA isolated.
These reagents are designed to provide lysis
and incubation conditions that reverse o A good preparation of plasmid DNA will yield
formaldehyde modification of RNA and a bright signal from supercoiled plasmid DNA
efficiently release RNA from tissue sections with minor or no other bands that represent
while avoiding further RNA degradation. nicked or broken plasmid