biolvi

NUCLEIC ACID
ISOLATION
Outline

General Steps in Nucleic Acid

Isolation

Purity Determination Nucleic Acid

Quantif ication
Why isolate nucleic acids?
• High quality DNA is obtained for
other applications/analyses
Examples:
• Restriction digestion
• Gene cloning
• Amplif ication
• DNA sequencing
Common Sources of DNA
• whole blood • epithelial cells
• hair • urine
• sperm • paper cards
• bones with collected
• nails sample
• tissues • bacterial
cultures
• blood stains
• fungal cultures
• saliva
• animal tissues
• buccal (cheek)
swabs • plants
General Steps in Nucleic Acid Isolation
1. Tissue Homogenization and Cell
Lysis
2. Denaturation and separation of
other biomolecules from the
nucleic acid
3. Precipitation of nucleic acid from
the aqueous phase
4. Washing of precipitated nucleic acid
5. Drying of pellet and dissolution of
dried pellet
N. Acid Isolation: General Steps
1. Tissue Homogenization and Cell
Lysis
• Mechanical method:
grinding,sonication

Bead-based Sonicatio
disruption n
N. Acid Isolation: General Steps
1. Tissue Homogenization and Cell
Lysis
• Mechanical method:
grinding, sonication
• Chemical method:
• buffer (e.g. Tris-HCl)
• salt (e.g. NaCl)
• cell lysis reagents (e.g. SDS)
• denaturants
(e.g. guanidine salts)
• Enzymatic method:
- lysozyme, cellulase,
N. Acid Isolation: General Steps
2. Denaturation and separation of other
biomolecules from the nucleic acid
• Chemical treatment
• phenol
– denatures proteins
• chloroform
– denatures proteins and lipids
• isoamyl alcohol
– reduces foaming and stabilizes
interface
N. Acid Isolation: General Steps
2. Denaturation and separation of
other biomolecules from the
nucleic acid
• Chemical treatment
• CTAB
(Cetyltrimethylammonium
bromide) – removes
polysaccharides
• PVP (polyvinylpyrrolidone) –
• Enzymatic treatment (e.g.
removes polyphenols
protease) Isaevde,
CC4.0

• Centrifugation
N. Acid Isolation: General Steps
3. Precipitation of nucleic
acid from the aqueous
phase
• •monovalent
sodium, cations
potassium, Squidonius,

ammonium
CC0

• •alcohol
ethanol
(95% to
absolute)
• isopropanol
• centrifugation Ipseidem,
CC4.0
N. Acid Isolation: General Steps
4. Washing of precipitated nucleic
acid

• 70% alcohol
• centrifugation
• may be done for two to
three times

Faunkhauser,
2001
N. Acid Isolation: General Steps
5. Drying of pellet and dissolution of dried pellet

• air drying or vacuum drying

• dissolution in sterilized molecular grade
water or TE (Tris-EDTA)
• EDTA – inactivates Dnases
Commercially-available
kits
After Isolation
• RNase treatment if DNA is isolated
• DNase treatment if RNA is isolated
• Storage:
• stock solution at -20˚ C
• working solution at 4˚ C
• Determination of purity and concentration
• UV spectrophotometry
• gel electrophoresis –
with DNA standards (different
concentrations)
Purity Determination
• Uses a UV Spectrophotometer
• Determine A260 and A280
• 260 nm – λmax for nucleic acids
• 280 nm – λmax for proteins

• Compute for A260/A280

• 1.8 – 2.0 : high purity of nucleic acid
isolate
• <1.8 (<1.6) : protein contamination
Purity Determination
• Another ratio used: A260/A230
• 230 nm – λmax for
• A /Acarbohydrates
260 23
•0 2.0-2.2 or > A260/A280 : pure isolate
• <2.0
• contaminated with carbohydrate carryover
(especially in plants), residual
phenol/guanidinium, other organic
compounds, salts
Purity Determination
Quantifying Nucleic Acids
• Use the ff. formula
• [ds DNA] μg/mL = A260 × 50 ×
DF
• [ss DNA] μg/mL = A260 × 37 ×
DF
• [RNA] μg/mL = A260 × 40 × DF
Quantifying Nucleic Acids
• Sample problem
• A 10 μL-aliquot of a resuspended genomic
DNA stock solution was obtained and
further diluted by adding 990 μL TE buffer.
The A260 of the resulting solution was 0.316.
Determine its concentration in μg/μL.

ANSWE
R:
[dsDNA] μg/mL =
0.316 × 50 × 100
= 1580 μg/mL or
= 1.580 μg/ μL
Gel Electrophoresis
• Movement of electrically charged molecules in
an electric f ield
• Separates molecules on the basis of net charge
and molecular weight
• The rate of migration of the molecules is
inversely proportional to their size with shorter
molecules moving faster and migrating farther
than longer ones.
Gel Electrophoresis
• For nucleic acids:
• Commonly used gel : agarose
• Visualizing agent : ethidium bromide,
GelRedTM
Gel Electrophoresis
• Please watch this video: https:
//www.youtube.com/watch?v=2UQIoYhOowM
Take note of the following:
- How do we compute the
amount of agarose needed
to make a gel?
- What is the effect of the gel
concentration on the rate of
migration?
- Why is the sample loading
buffer (which contains dyes
and glycerol) added to the
sample prior to loading?
Staining with Ethidium Bromide
• Ethidium bromide
• Intercalating agent
• UV absorbed by DNA at
260 nm is transmitted to
the dye
• EtBr in UV light emits a
red-orange color
(f luorescence) at 590
nm detected by the
naked eye
Visualizing EtBr-stained Gels
• UV Transilluminator and Gel
Documentation System
Gel of DNA samples with
molecular weight ladder
Gel of DNA samples with DNA standards
Λ DNA
2000 1000 800 500 100 MW ??? ??? ??? ???
ng ng ng ng ng ladder
Why do absorbance-based and gel electrophoresis-
based quality checking?

• Absorbance-based detection
• Cannot discriminate DNA and
RNA
• May lead to overestimation
of concentration
Other methods of determining concentration and
purity
Fluorometer
(e.g. Qubit®, Life
Technologies)
- uses f luorescent dyes to determine the
concentration of nucleic acids and proteins in a
sample
https://www.thermof isher.com

Microf luidics
(e.g. Agilent Bioanalyzer, Biorad Experion)
- On-chip gel electrophoresis

BioRad
Suggested Online Activity:

Virtual DNA Isolation:

http:
//learn.genetics.utah.edu/content/labs/extraction/
End of NA
Isolation

next topic…
DNA denaturation and
renaturation
Reference
s:
Barbosa C, Nogueira S, Gadanho M, Chaves S. 2016 Molecular Microbial
Diagnostic Methods: Pathways to Implementation for the Food and
Water Industries http://dx.doi.org/10.1016/B978-0-12-416999-9.00007
-1
Huma
Extraction and Precipitation of DNA. (2001). Current Protocols in n
Genetics. doi:10.1002/0471142905.hga03cs00
Fankhauser. D 2001. Isolation of Buccal Cell DNA . Retrieved July 2020. Available
from https://fankhauserblog.wordpress.com/2001/01/19/isolation-of-buccal-
cell-dna/

Sirakov IV. 2016. Nucleic Acid Isolation and Downstream Applications, Nucleic
Acids - From Basic Aspects to Laboratory Tools, Marcelo L. Larramendy and
Sonia Soloneski, IntechOpen, DOI: 10.5772/61833. Available from: https:
//www.intechopen.com/books/nucleic-acids-from-basic-aspects-to-
laboratory- tools/nucleic-acid-isolation-and-downstream-applications

*Some figures from wikimedia creative commons