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Molecular Biology and Diagnostic

Laboratory: DNA Isolation
By: Adlene Atienza
I. Preparation of a cell extract
II. Purification of DNA from cell extract
III. Concentration of DNA samples
DNA Extraction IV. Measurement of purity of DNA
concentration
❖ DNA extraction is a procedure used to isolate
DNA from the nucleus of cells from other
components
I. Preparation of Cell Extract❖
Preparation of Cell Extract
❖ Process of purification of DNA from various o To extract DNA from a tissue/cells o
sources using a combination of physical and interest, the cells have to be separated
chemical methods and the cell membranes have to be
❖ Methods used to isolate DNA are dependent on disrupted
o The “Extraction buffer”, EDTA and SDS
the source, age and size of the sample helps in carrying out these processes
Purpose of DNA Extraction o Having lysed the cells, the final step in
the preparation of a cell extract is
❖ To obtain DNA in a relatively purified for which removal of insoluble cell debris
can be used for further investigation ❖ DNA Extraction from Whole Blood Sample
o Scientific
o Ficoll-Directed Gradient Through
o Medical
Centrifugation
o Forensic purposes
o Proteinase K
o Example:
o Phenol
▪ PCR

▪ Sequencing

Sources of DNA for Molecular

Biology ❖ It can be isolated from any living
or dead organism
❖ Common sources for DNA isolation:
o Whole blood
o Hair
o Sperm ❖ DNA Extraction from Dry Blood Spots
o Bones o Chelex-1000
o Nails o InstaGene Matrix
o Tissues
o Blood
o Stains
o Saliva
o Buccal swabs
o Epithelial cells
o Urine

Process of DNA Extraction

❖ Isolation of DNA basically consists of four major
sites
o
Notes by: Jarrylle Guzman//2MT-P

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▪ Heat and Microwave

▪ Heat and Mineral oil

o Digestion
▪ Proteinase K
o Purification
▪ Phenol/Chloroform + Alcohol

▪ Silica-based
o
▪ Salting Out
❖ Non-Invasive Human DNA Isolation
o Hair II. Purification of DNA from Cell
▪ Alkaline Lysis Method Extract
❖ In addition to the DNA the cell extract will
▪ Digestion
contain significant quantities of detergents,
o Saliva proteins, salts and reagents used during cell
▪ Buccal Swab lysis step and RNA
❖ A variety of procedure can be used to remove
▪ Mouthwash Method
these contaminants, leaving the DNA in a pure
o Urine form
❖ DNA Preparation from microorganisms o ❖ Cell debris and partially digested organelles can
Bacteria and Fungi have cell walls that must be be pelleted by centrifugation leaving the cell extract
broken to release the nucleic as a reasonably clear supernatant.
acid
o Physical methods and chemical ❖ The most commonly used procedure are:
methods may be used o Phenol-chloroform extraction
o Chemical Method: o Inorganic DNA extraction
o Minicolumn purification
▪ Detergents
❖ Organic Extraction Reagents:
▪ Lysozyme
o Cell lysis Buffer
▪ Lyticase o EDTA
o Proteinase K
▪ CTAB o Phenol/ chloroform-remove
proteinaceous material
▪ Chelex-100 o TE Buffer
❖ DNA Extraction from Formalin-Fixed, Paraffin o Ethanol
Embedded Tissue ❖ Organic Extraction:
o Deparaffinization
o Glycine in a alkaline environment

o De-waxing
▪ Heat o The buffer will lyse the cell membrane
o The nuclei is resuspended in a buffer
▪ Organic solvents containing SDS (Sodium Dodecyl
Sulfate) and Proteinase K
 o DNA release into solution is extracted
❖ Inorganic DNA Extraction
with phenol-chloroform DNA
o DNA is precipitated from the aqueous o Does not use organic reagents such as
layer by the addition of ice ethanol and phenol or chloroform
alt o Digested proteins are removed by
o Precipitated DNA is washed with 70% salting out
ethanol, o However, if salts are not adequately
removed, problems could occur with the
▪ Dried under vacuum and
resuspended in TE buffer

Notes by: Jarrylle Guzman//2MT-P

Helping Hand

procedure due to alteration of DNA

mobility (band shifting)

o Lysis
▪ The cells of a sample are
broken with a lysis procedure
o Binding
o Cell lysis buffer containing proteinase— ▪ Buffer solution is then added to
lyse cell membrane, lyse nuclear the sample along with ethanol or
membrane and digest protein at high isopropanol.
temperature
o To remove proteinaceous material • This forms a binding
solution
▪ LiCl is added and incubated on o Washing (3x)
ice
o DNA remains in solution ▪ The flow-through is removed

▪ Transfer supernatant to a new ▪ Then wash buffer is added to

tube, care must be taken not to the column
take any of protein pellet o Drying
o DNA is precipitated by the addition of
▪ The wash buffer is removed
room temperature isopropanol,
▪ LiCl will precipitate with DNA ▪ Then elution buffer (water) is
o Precipitated DNA is washed with 70% added to the column
ethanol and resuspended in TE buffer o Elution

❖ Minicolumns Purification ▪ The elution buffer removes the

o Relies on the fact that the nuclei acid nucleic acid from the membrane
may bind (thru absorption) to the solid and the nucleic acid is collected
phase (like silica o other) depending on from the bottom of the column
the pH and the salt concentration of the ❖ Enzymes, Reagents, Chemicals
buffer
o A spin column using a silica-based Enzyme/Reagent/Chemical Purpose/Function
extraction method is used
▪ This does not require the use of RNAase (ThermoFisher) Degrades
single-stranded RNA
hazardous chemicals
o Nuclei acids are attached to the silica Buffer 1 Dissolves RNAse
bead under high chaotropic salt
concentrations
 Lysozyme (sigma) Lyse Gram-negativee
bacterial cell wall
III. Concentration of DNA Samples ❖ Most
Achromopeptidase (sigma) Lysa Gram-positive
bacterial cell wall frequently used method of concentration is ethanol
precipitation
Sodium dodecyl Solubilize cell ❖ With a concentrates solution of DNA one can
sulfate (SDS) membrane lipids
use a glass rod to pull out the adhering DNA
strands
Proteinase K Digest proteins
(ThermoFisher) ❖ For dilute solution, precipitated DNA can be
collected by centrifugation and re-dissolving in
Phenol: Chloroform: Separates DNA from an appropriate volume of water
Isoanyl Alcohol other cellular materials
IV. Measurement of purity of DNA
Ethanol Precipitates DNA concentration
from Solution
❖ DNA concentration can be accurately measure b
Tris-EDTA Dissolves precipitated UV absorbance spectrometry
and dried DNA

Notes by: Jarrylle Guzman//2MT-P

Helping Hand
Spin the tube at 10,000 rpm for 5 minutes 14.
Discard the supernatant by pipetting out the
❖ The amount of UV radiation absorbed by a alcohol
solution of DNA is directly proportional to the a. The, rinse the DNA with 70% ethanol
amount of DNA samples 15. Spin as before and carefully pipet out the
alcohol
❖ With a pure sample of DNA the ration of the
a. Then, Air dry the bacterial DNA
absorbance at 260 nm and 280 nm (A260/A280)
is 1.8
RNA Isolation
Isolation of Genomic DNA ❖ RNA (ribonucleic acid) is a polymeric substance

from Bacteria (Classical) 1. present in living cells and many viruses

Pick 3 similar colonies from the Luria-Bertani ❖ RNA is only a single long strand of nucleotides ❖
Agar plate and inoculate in 5mL LB broth It is used in all steps of protein synthesis in all living
(incubate) cells ad carries the genetic information for many
2. Pipette 1.5 ml of the broth culture into an viruses
Eppendorf tube
a. Then, spin at 10,000 rpm for 5 minutes Purification of RNA from Biological Samples
3. Remove as much supernatant ❖ The isolation of RNA with high quality is a crucial
4. Add 500 ul lysis buffer and vortex for 1-2
minutes to resuspend bacterial cells step required to perform various molecular biology
5. Incubate for 1hour at 37 ˚C experiment
a. Then, add 600 ul phenol:chloroform (1:1 ❖ This procedure is complicated in the presence of
volume) ribonuclease enzymes in cells and tissues
6. Spin at 10,000 rpm for 5 minutes
7. Transfer the supernatant to another Eppendorf ❖ Norther Blot Analysis, molecular cloning in vitro
tube translation
8. Repeat the incubation and phenol: chloroform
extraction at least 4x Ribonucleases (RNases)
9. To finally remove the phenol, add to the
aqueous extract an equal volume of chloroform 10. ❖ Are naturally occurring enzymes that degrade
Spin at 10,000 rpm for 5 minutes RNAs
a. Then, remove the supernatant to a new
❖ Common laboratory contaminant
Eppendorf tube
11. Add 2.5-3.0 volumes of cold 100% ethanol ❖ Also release from cellular components during
12. Place Eppendorf at -20 C for 30 minutes 13.
 isolation of RNA from biological samples ❖ ❖ Denature nucleic acid -protein complexes
Difficult to inactive
❖ RNA selectively partitioned from DNA and
Inhibitors of RNase protein

❖ DEPC (diethylpyrocarbonate) How to avoid Contamination

o Alkylating agent ❖ Contaminated Solutions/Buffers
▪ Modifying proteins and nucleic o Use good sterile techniques
acids o Treat solution with DEPC
o Make small batches of solution
❖ Vanadyl ribonucleoside complexes
o Competitive inhibitors of RNAse ❖ Contaminated equipment
o Use RNA-only pipettes, glassware
❖ Protein inhibitors of RNases
o Maintain a separate area for RNA work
o Horseshoe-shaped, leucine rich protein,
found in cytoplasm of most mammalian Basic Steps in Isolating RNA from Clinical
tissue Specimens
Precautions for Working with RNA in the ❖ Cell Lysis
Clinical Laboratory
❖ Denature/digest proteins
❖ Use Rnase-free tubes and pipette tips ❖
❖ Separate proteins, DNA and contamination from
Always wear gloves and work in a hood ❖
RNA
Treat liquids with DEPC
❖ Precipitate RNA if necessary
❖ Cells or tissue must be rapidly and efficiently
disrupted ❖ Resuspend RNA in final buffer

Notes by: Jarrylle Guzman//2MT-P

Helping Hand
RNA Extraction Methods

Cell Lysis and dissolution Buffers or reagents

containing chaotropic
agents (guanidium
isothiocyanate,
guanidium chloride,
SDS, TRIzol)

Denaturation of DNA DNase, proteinase K,

and Proteins phenol and
chloroform, buffers
with guanidium salts

Denaturation and Chaotropic agents,

inactivation of RNases phenol and chloroform

Separation of cellular Chloroform and

components centrifugation

Precipitation Isopropyl alcohol,

ammonium acetate
 Filter-Based RNA Isolation
❖ Filter-based formats utilize membranes that are
seated at the bottom of a small plastic basket
❖ Samples are lysed in a buffer that contains
RNase inhibitors are bound to the membrane by
passing the lysate through the membrane using
centrifuge force
❖ Wash solutions are passed through the
membrane and discarded
❖ Elution is applied and the sample is collected
into a tube by centrifugation

Qualification of Isolated RNA

❖ UV Spectroscopy
o The A260/A280 ratio is used to asses
RNA purity
o A A260/A280 ratio of 2.0 indicates
highly purified RNA
Organic Extraction Method
❖ Phenol/guanidium solution disrupts cells
,solubilizes cell components, but maintains the
integrity of RNA
❖ Add chloroform, mix and centrifuge

❖ Samples separates into 3 phases:

o Lower organic phase
o Middle phase with denatured proteins,
DNA
o Upper aqueous phase contains RNA
❖ RNA is collected by alcohol precipitation

TRIZOL RNA Isolation Protocol

❖ TRIzol Reagent is a ready-to-use reagent used
for RNA isolation from cells and issues
❖ It works by maintaining RNA integrity during
tissue homogenization
o While at the same time disrupting and
breaking down cells and cell
components
❖ Light sensitive and often stored in a dark
colored, glass container covered in foil
❖ TRI is trizole stands for total RNA isolation
o It is a ready to use reagent contain
phenol, guanidinium salts, red dye and
other components for the isolation of
RNA in a simple step process

Inorganic Salt Precipitation

❖ Cell membranes are lysed and proteins are
denatured by detergent in the presence of EDTA
or other RNase inhibitors
 ❖ Proteins/DNA are precipitated with a high
concentration salt solution
❖ RNA is precipitate with alcohol and rehydrated

Notes by: Jarrylle Guzman//2MT-P