Molecular Biology

LECTURE WEEK 4: DNA & RNA ISOLATION TECHNIQUES

NUCLEIC ACID ISOLATION TECHNIQUE  we use blood specimen to isolate but we

GENERAL OVERVIEW make sure to collect particularly
 The purpose of extraction is to lymphocytes
release the nucleic acid from the cell
for use in subsequent procedures. A.Differential density gradient centrifugation
 The goal is to isolate a nucleic acid 
★Sample: Mononuclear cells of
that is free of contamination and
WBC
damage.
★ Principle:  Uses Ficoll-Paque that is
made up sucrose
Two Types of Nucleic Acid
polymers that does not
1. DNA penetrate biological
2. RNA membranes and able to
separate substances
 In molecular level, these are being based on density
targeted, we want them to be extracted (Centrifugation)
to facilitate or gather their sequences.  When we say density,
 Usually, the purpose of isolation is to we are talking about the
simply release the nucleic acids coming specific gravity. For
from your sensors and that would be gradient centrifugation,
used for further testing.
gradient is we are to add
 In short, our main goal is to get the
another substance that
nucleic acid, and we all know on a basic
will provide the gradient
part of the cell going back to histology:
material so we make use
 DNA has cell wall, nucleus, organelle
inside. Within our nucleus, you will find
of what we call sucrose
the nucleic acid DNA and RNA. Para polymer or Ficoll-paque
makuha natin yan, we have to make kumbaga siya yung
sure na madestroy the entire cell if we parang nag-facilitate to
are after the nucleus to isolate NUCLEIC separate better
ACIDS. Note: This is for the first part, wala tayong
sisirain, only separate them.
According sa isolation steps, we have for DNA and
for RNA. B.Differential lysis
★ Sample:  Mononuclear cells of
WBC
DEOXYRIBONUCLEIC ACID ISOLATION (DNA)
★ Principle:  Involves incubation in
SPECIMEN PREPARATION METHODS hypotonic buffer or
 this could be blood or microorganism water to initiated lysis to
 We have pre-treatment procedures prior to release nucleus
extraction  Because the goal is to
1. Nucleated cells lyse agad the cells
 mononuclear cells is a type of cell that Note: Problem with differential lysis is we have
has one nucleus (lymphocyte), in which many contaminants (organelles) kasi sinira lang
appearance has a higher nucleus content natin yung nag-contain sakanila or the cell wall
compared to cytoplasm. so madami pa din nakikita sa solution natin. To

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Transcriber: LCB
Molecular Biology
LABORATORY WEEK 2: MOLECULAR CLINICAL LABORATORY

remove, that would be on a different procedure
na.
2.Tissue Sample
 our tissue is filled with many nucleus, from
histology we have cuboidal cells (many
nucleus), we have squamous, and
transitional epithelium.
★ Sample:  Fresh, fresh frozen, Fresh
Usually from frozen-paraffin embedded
Histopath lab tissues (FFPE)
 the basic sequence: cell
-> tissue -> organs
★ Principle:  Grinding of hardened
tissues by using liquid
nitrogen,
 or thru homogenization,
or simply mincing helps in
the release of nucleic
material to be isolated (for
fresh and frozen);
 For embedded samples
must be deparaffinized
first by soaking to xylene
and rehydrate to
decreasing concentrations
of ethanol
3. Microorganism
★ Sample:  Bacterial isolates
 these are prokaryotes
★ Principle: Bacterial cell wall must be
broken to release the nucleic
substances
A. Mechanical treatment
 grinding or vigorous mixing with glass
beads
 glass beads will get or absorb the nucleus
(for vigorous mixing but sa grinding
walang glass beads)
B. Chemical treatment
 best choice for bacterial
 enzymatic method with less likely
damages
 Uses 1% sodium dodecyl sulfate (SDS)
and 0.2M NaOH in the presence of Tris-
base, EDTA and glucose to break the cell
wall
 DNA can be released by means of boiling
with 8% sucrose, 8% triton X-100, Tris-
buffer and EDTA after lysozyme
treatment releases DNA and must be
precipitated with alcohol
Purification Method
 after isolation of nucleic acids, we have to
purify to remove contaminants
1. Organic Isolation Methods
 organic because we will make use of
organic substances such as phenol,
and chloroform para maiwan nalang si
DNA
★ Goal: Dissolve hydrophobic contaminations
and strips away DNA associated
proteins
 Uses high salt, low pH, and organic
material such as phenol and
chloroform to remove contaminations;
 DNA is isolated and precipitated using
ethanol or isopropanol in high salt
concentration leading to formation of
solid precipitate;
 precipitated DNA is collected by
centrifugation and excess salt is
removed by rinsing the pellet in 70%
ethanol
 Phenol and chloroform create biphasic
emulsion when added to hydrophilic cell
lysate and DNA is precipitated using
ethanol in high concentration of salt and
thru centrifugation to settle the
hydrophobic and hydrophilic layer
 ★ Fungal sample is facilitated by
pretreatment with
cetyltrimethylammonium bromide
(CTAB) to efficiently separate DNA from
contamination

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Transcriber: LCB
Molecular Biology
LABORATORY WEEK 2: MOLECULAR CLINICAL LABORATORY

Step 1: Specimen preparation

Step 2: Cell lysis

Step 3: Precipitation (nasa bottom ang

debris, sa taas ang dna)

3. Solid Phase Isolation

★ Goal: bind DNA from contamination
solution
Step 1: Specimen preparation  Ideally used for viral and bacterial DNA
isolation from serum/plasma or CSF
Step 2: Cell lysis specimen
Step 3: Acidification - ipapagsettle lahat ng  Rapid and effective method; uses silica-
cell debris sa bottom based products than can bind DNA in high
salt conditions thru spin column
Step 4: Washing - dito na gagamitin si phenol centrifugation and washed with buffer;
and chloroform and will create biphasic eluted in specific volume of water, TE or
emulsion (or layers) other low salt buffer and washing solution
Step 5: DNA Precipitation - transfer the can be drawn through the column by
supernatant that contains Dna and we gravity, vacuum or centrifugal force
will make use of ethanol / isopropanol
for visualization of the DNA para mag
settle down na ulit. (Buffer and DNA
for storage)

2.Inorganic Isolation Methods

★ Goal: remove protein by selective
precipitation
 Known as “salting out”;
 uses low pH and high salt condition;
DNA is isolated and precipitated using
isopropanol pelleted and resuspended
in TE buffer or water
Step 1: Specimen preparation

Step 2: Acidification para Bumaba ang mga

debris
Step 3: Spin column centrifugation and
washing (ma-wash lang ang mga
hindi dna; didikit sa silica ang dna)
Step 4: Elution - release the Dna from silica

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Transcriber: LCB
Molecular Biology
LABORATORY WEEK 2: MOLECULAR CLINICAL LABORATORY

4. Crude Lysis 1.Total RNA Isolation

 concerned towards the properties of  80 to 90% rRNA
cells especially mga galing from paraffin- ★ Specimen: reticulocytes
embedded (centrifugation/lysis), tissues
A. Proteolytic lysis of fixed material (liquid nitrogen), microorganism
★ Sample: paraffin embedded samples (mechanical or chemical)
★ Detergents: SDS or triton, Tris-buffer and ★ Detergents: guanidine thiocyanate
proteinase K (denaturant) and 2-mercapthoethanol (reducer)
B. Extraction with chelating resin ★ Principle: Lysis is done by using
detergent or phenol in high salt
★ Sample: FFPE, Forensic specimen
concentration and RNA is
★ Detergents: 10% Chelex is a cation- precipitated using 70% ethanol
chelating resin used for and resuspended in buffer or
simple extraction water
 The sample is chelated, boiled and  Protein is removed by
centrifugated; DNA is separated thru extracting with phenol
chloroform extraction before following the acid
amplification steps phenol- chloroform-
isoamyl concentration
of 25:24:1 respectively
 Chloroform
(carcinogenic) enhances
extraction by denaturing
proteins and promoting
phase and isoamyl
prevents foaming

RIBONUCLEIC ACID ISOLATION (RNA)

Initial preparation
 RNA is especially labile due to the
ubiquitous presence of RNAses and must
be inactivated
Step 1: Specimen preparation
 Laboratory precautions is strictly followed
to avoid damage to isolated RNA
samples Step 2: Cell lysis (use of guanidine
thiocyanate (denaturant) or 2-
mercapthoethanol (reducer)
Methods of Isolation

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Transcriber: LCB
Molecular Biology
LABORATORY WEEK 2: MOLECULAR CLINICAL LABORATORY

Step 3: extraction with help of P-C-I to remove

contaminants
Step 4: precipitated rna (ethanol)

2. mRNA Isolation
 2.5 to 5% only
 where all genetic material is found
 to isolate, we have to employ solid-phase
isolation
 Uses matrix resin column or beads for
better yield of mRNA and some oligomers.
 Involves the attachment of polyT & polyU
to polyA

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Transcriber: LCB