Isaac O.

Obonyo
lectures@a020/2021
The
extraction of ntroduction to Biomolecules Extraction in
molecular biology.biomolecules,
method used
crucial
most
is the
It is the DNA, NA, and piprotein,
RNA, and development

including diagnostic kits. starting point tor

for downstream
processes
downs
and product
any
biological
material DNA, RNA,
from

and protein an
isolated
DNA.D be
be
such as
living or Conser and
procells,
can
1s other
other samples
for
analytical preparativ
or
preparative uposes, conserved tissues, C
SSues, paru
virus particles,
virus
Or
or

Two purposes.
categories
constructs
that
involved in the isolation
of
recombinant DNA
such as
plasmids or purifying
ying DNA include
DNA or genomic

isolation O
chromosomal
DNA from isolation of
prokaryotic bacteriophage
or eukarvo ophage and the
and

Generally, successful eukaryotic organisms.

Of
etfective
steps:
disruption of cells nucleic acid
important
or purification required four
purification
requtein
nucleases, for example,tissue;
RNase denaturation
for RNA cxtraction
of ree compie
nucicat 1 DNase offorcontamia
DNA traction;
from away
contamination. The contaminants including

ydrate, lipide
target ucleic
nucleic acid
acid
shou
should be free
of
free of RNA or

RNA fro
RNA free of DNA. other nucleic acid, for example,example, DN DNA

Quality and also integrity of directly affect the

results of all
the isolated nucleic
15olated acid will
nucleic acia
Succeeding scientific research. On the other unstable moleculea has a
very short half-life once hand,
RNA
extracted from the cell or tissues. are everal types
of urally
naturdi
OcCurring RNA including ribosomal tn
RNA (rRNA) (80%-90%), messeu
(2.5%-5%) and transfer RNA (tRNA).
pe Care and precautions are required for it is susceptible to
RNA isolation as

ridaon. KNA 1s especiallyunstable.due to the ubiquitous presence of RNases which

are

Cnzymes present in blood, all tissues, as.well as most bacteria and fungi in the environment.
Sirong denaturants have always been used in intact RNA isolation to inhibit endogenous
RNases RNA extraction relies on good laboratory technique and RNase-free technique.
RNASe is heat-stable and refoldsfollowing heat denaturation. They are difficult to inactivate
as they do not require cofactors. The most common isolation methods can be divided into two
classes: utilization of 4 M guanidinium thiocyanate and utilization of phenol and SDS.

DNAExtraction/PurificationBasics
Basic Isolation Procedure

There are five basic steps of DNA extraction that are consistent across all the possible DNA

purification methods:

cellular structure to create a lysate,

() Disruption of the
(2) Separation of the soluble DNA from cell debris and other insoluble material

of interest to a purification matrix,

the DNA
(3) Binding
 Isaac O.
Obonyo
(4)
lectures@2020/a021
DNA.Washing proteins and other matrix
and 5)
elution
of the

1.
Creation of Lysate contaminants
ants away
from the

The first into

step in
any
DNA/RNA

solution. The goal of nucleic acid reaction is releasing

the
to
release

nucleic acid into

the
lysis is to iication
rapidly
methods, enzymatic lysate. There are four and purificationetcly
disrupt
completely
for
cells in a
sample
materials:
physical

methods, chemiOur
Ods, chemical
and
comechniquens
general

geneicombina
for lysing
y
techniques
of the three.

Physical Methods methods a

Physical methods to disrupt
the

ell walls or tough typically involve esome type

or crushing
grinding grinding
type of sample is freezing
and

tissue. Acommon method o ical disru

disruption that is
samples with a mortar ethod of physical
of material

pestle under liquid puyto

powdered
and pestle
and Drovide a n be s
to provide
a be simple manual

then exposed to chemical nitroBns. ods are

Grinae
e n z v m o : d n i t r o g e n

devices or automated, capableOr or enzymatic lysis Physical metho

con
of disruption of mui 6-well pla ices use
often used with more
structured input materials, a stissues p
t 1sst or other dev
cells or
bead beating or
shaking su disrupt
to disrupt
beads to
Co

tissues, or sonication to in the presence of meta

ceramic

disrupt tissues and lyse

cells
Chemical Methods
culture cells
Chemical methods can be used alone such as tissue
with easy-to-lyse materials,
O combination with other methods, Cellular disruption is accomplished wi d
used
Chemicals commonly
agents that disrupt membranes and denatures proteins. and alkaline solutions).
Include detergents
(e.g., SDS) and chaotropes (e.g., guanidine salts and as
Enzymatic Methods
materials in combinaton
Enzymatic methods are often used with more structured starting
bacteria and yeast. The enzymes utilized
With other methods with tissues, plant materials, on the starting material, typical
help to disrupt tissues and tough cell walls. Depending
and liticase, proteinase K, collagenase
enzymatic treatments can include: lysozyme, zymolase
treatments can be amenable to high throughput
and lipase, among others. Enzymatic
to other disruption methods.
compared
processing, but may have a higher per sample
cost

of chemical disruption and another is often used since

NB-In many protocols, a combination inactivates proteins, including nucleases.
chemical disruption of cells rapidly

2. Clearing of Lysate
may need to have cellular debris removed
Depending on the starting material,tocenular ysarcs
of unwanted materials (proteins.
prior to nucleic acid purification
reuuce
tnc cayover ns,
charides from cellular structures nto tne plurilication reaction, which can clog

membranes or
interfere vith downstream applications. Usually clearing is accomplished by
filtration or
bead-based methods.
centrifugation,
time, but it is able to
require
more hands-on S
arge amounts of
Centrifugation can
samples with
samples laroe
d method,
methoa, but With aa
large amount of debris can clog
debris. Filtering can be a rapid
 Isaac O.
Obonyo
the
filter.
lectures@2020/a031 plasmid

Bead-based
prep kits, can
particle-based
a

cleared lysate beis used inclearing,

Once
Promega b i o m a s s .

the like used

with
overwhelmed
with such

metnocan
chemistries,

as
silica, 1on generated, automated
the DNA protocols,
Proo
od
but can
be d i f f e r e n t

exchange,
many

3.
Binding to the
ge,
he
cellulose
ulosA

or
Can
can then
C n be
precipitatio
precipitation-base
urificthods
be
purified
by
ne

Regardless of the
Purification Matrix can
be
isolated

using a
on variety of method used to create lysate,
the DNA
of
interest

systems
based

different
i s o l a t i o n

and
sample lysis by methods.
D
a cleared

cleaners genomic
DNA

(silica,
cellulose

Promega
1on
exchange), whichdetergents, gettomacent
ctergecthods offers matrices

to

on by bindinscd
and puriga
is
where
SWhere
dnd
purification binding
in
Each of these nterest
prima heenandfou purity of the
interest hhas each
and
isolation,

have a chemistries can influence the

acid an characteristic binding influence
Can efficiency
the efficiency andication o syster
isolation
C

capacity.
a p

Bind ennety is an nacity

a e t h e

stem ne
and no

longer isolates morechemistry can bind before caphes the 1t

apa
Othese Ch into these
c h e m i s t r i e s

by of that nucleic acid. We d design

features
(e.g
manipulating the binding build
can for
design
w e can

fferent categories
of
nucleic
acid

chemistries that
selectively conditions O enncn large
to enrich Cadifferent
all fragnm Sma
bind RNA
Cy bind RNA versus DNAver
Solution-Based Chemistry
This type of chemistry does rather on
alcohol
precipitation.

not
Following the creation of lysaterely
t binding matrix, but are precipitated
using a high-
On of lysate, the cell debris and proteins
debris and
f
the proteins
to fall out
of
thOlution. The hi of salt causes

debris and
Solution,and centrifugation
Concentration
nucleic acid
from the cell
precipitat c parates the soluble
precipitated protein.
N A 1 s then precipitated by adding isopropanol to the high-concentrauo solution.
while the smaller RNA
Bgenomic DNA molecules out of solution, irom s l s
fragments remain soluble. The insoluble DNA is then peileted and separated
isopropanoland RNA
fragments via centrifugation.
Additional washing of the pellet with ethanol removes the remaining salt and enhances
evaporation. Lastly, the DNA pellet is resuspended in an aqueous buffer like Tris-EDTA
or

nuclease-free water and, once dissolved, is ready for use in downstream applications.

Silica-Binding Chemistry

The technology for these genomic DNA purification systems is based binding of the DNA
tosilica under high-salt conditions (2-4),. The key to isolating any nucleic acid with silica is
on

the presence of a chaotropic salt like guanidine hydrochloride. Chaotropic salts present in
high quantities are able to disrupt cells, deactuvate nucieases and allow nucleic acid to bind to

silica.
Once the genomic DNA is bound to the Silica membrane, the nucleic acid is washed with a
salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and
keeping the DNA bound to thes
Once
small the
RNAs to increase
washes purity while
are finished, the genomic DNA is eluted under 1low-salt
embrane
conditions
column
using

water or 1E buffer.
nuclease-free
either
 Isaac O.
Obonyo
Binding to silica is
lectures@2020
add
gDNA,ribonuclease
the optioni
not DNA there is also
with

and the (RNase specific, required, copurified

majority of addition ofA) o the elution pu so if

elut:Pure
DNA is
may
be may
be
o f the
vast

contaminating RNA.RNaseSe toto Cthe eDon

removal
RNA
Rhe buffer. the

Cellulose-Binding Chemistry
ensures
natinf
buffer
bu

elution

More
based recently, Promega has
matrix. Nucleic that
use a
cellulose-

Generally
methods

commercialized
alcohols.

isolation
salt a n d
acid DNA
can be speaking, hthe
bin binds tlized
d s to presence
of high Conditions

the adjusted
sted to to
e
bind capacity cellulos
ose
in the
the
metho methods
is very high. result of

combination ofpreferential y bind baseand acid. As

a
of cellulose-based

concentration eluatesbinding ind celluies and sizes

of nucleic
get high
capacity different
we can
species
Cnt S elution
volume,
for pacity and small
relatively sma
lon Exchange Chemistry nucleic
CiC acide
acids. rela
lon
exchange chemistry is
particles and the based on the that occurs
between
positively-charged

DNA binds
under loW dlt
salt conditi negatively-charged
charoed
With higher salt conditions, and
nteraction

phosphatesns
Osphates
that present in
DNA. The
are

can then
be washed away

by ethanol precipitation. contaminating

solutions. The DNA proteins
ng pro and RNA
RNtio
lhe DNA is eluted under high
and
conditions, and
salt conai
h salt
then recovered

CIuted undc
4. Washing

Wash buffers
generally cacontainonols
alcohols and can be used
can be to remove proteins, salts and other
eninants from the us buffers. Alcohols additionally help
associate nucleic
acid with the tne upstream binding
5. Elution
matrix.

DNA 1S soluble in low-ionic-strength solution such as TE buffer or nuclease-tree

watet
wnc Sucn an aqueous buffer is.applied to a silica membrane, the DNA is released trom the
Silica, and the eluate is collected. The purified, high-quality DNA is then ready to use in a
wide variety of demanding downstream applications, such as multiplex PCR, coupled in vitro
transcription/translation systems, transfection and sequencing reactions.
When selecting your elution butfer, it is important to consider the requirements of your
desired downstream processes. Eluting and storing the DNA 1n TE buffer, for example, is
helpful as long as the EDTA does not impact your chosen downstream applications. EDTA
chelates, or binds, magnesium present in tne purnied DNA and can help inhibit possible
contaminating nuclease activity. If EDTA 15 a concern, we recommend storing DNA in a
buffered solution, as the acidic nature or DNA can lead to autohydrolysis. Alter
can use TE-4 buffer, which is 10mm Iris-HCI, 0.Imm EDTA (pH 8.0
 1saac O.
Obonyo
lectures@a020/a021
1.Scientiic use-DNA and
plants
to

generate Extraction a n i m a l s

urpOses oforrDNA
DNAD
v a r i o u s

of
has of
defects andgenetically
diseases. d number of
modification
p u r p o s e s

Dutyk
a

modified organisms(appl an in d i a g n o s t i c

Sanismeications
also for

2.Medicine- Is the most

extraction
GMO)
and

is
applied.
In
outbreaks
this
field,

and

of DNA e x t r a c t i o n

to
predict virulence isof used common
community-based
field where
DNA

point sourceswuhosp
to
POlnt
Sou
hospital
and

3.Forensic mi croorganisms. (for

Tor
1o S
i n d i v i d u a l s

science-ln
example rapists, this
i d e n t i f i c a t i o n
of

thieves, field DNA

for
is
extracoater
determination)

accidents/war victims
Onts/warCtraction

paternity
ums and

Phenol: chloroform method RNA extraction

This procedure is Guanidinium
thiocyanate

(GTC), based on the detergent GTC is

followed organic sample
in cationic Moreover,

effective at by
inactivating endogenous
ysis
lys1salcohol
extractions alcohol
precipitation.

Pbetter removal
of DNA
from

partof
the

aqueous phase, addition of low-pH ribonucic

easily made mixtures phenol
ces. For
leases. For
mmendeded, and
is
removahecomeilable
thus it
commercially
available

reagents like TRIZOL pHsuchof about

as
as
original "Solution DasO well n

4.
is
Chloroform is a purely The solution
the cell lysate.
organic solvent, unable
unable to mix
ni
with
properly interspersed, and then vent,
the enables effective
separation of uppct tne

Pdse and "lower nha u ne centrifugation

centrifugation enabi chloroform, respectively. At the

ase ntaining the cell lysate and

which contains
interface, a
ring of precipitated proteins appears. The "upper phase",
white
reaction with
sterile tube, where it undergoes the
rcd into a new test
a Wnite peiict
Sopropanol resulting in precipitation of RNA. After further centrifugation,
representing total RNA is recovered.

This characteristic white colouring is due to the presence of residual salts precipitating
the pellet using ethanol
together with RNA. Such salts are removed by washing
(concentration varies from 75-90 % according to the manual instructions).
Pure RNA is then dissolved in RNase-free water or in an appropriate buffer.

One of the main drawbacks of this principle may be the

incomplete dissociation of
nucleoproteins from the RNA. 1o avod t s , tne upper phase after isopropanol addition
should be kept a t room temperature Tor d TCw n u t e s Delore the consequent centrifugation

Cross-contamination of hoth "upper". and "lower" phases represents another possible

separation obstacle.

when transterring aqueous phase

tne aqueous phase to the
the fresh
fresh
tube has to be
avoided.
Carryover technicalities,
the phenol: chloroform merk
ESents the
Irrespective of these
recent
"in-lab" mdividually designed protoe
ocols.
standard, namely
for routine
Isaac O.
Obonyo
lectures@2020/2021
Isolation
such as
of intact RNA Furposes for RNA EX ction analysis
expression
is
essential fornany
many
techniques
used in gene

Microarray analysis
Northern blot analysis
CDNA library construction
RT-PCR