UNIT 4: NUCLEIC ACID ISOLATION

1. Lysis

2. Removal of unneeded biomolecules

3. Precipitation of nucleic acids

General flow of NA Isolation (3 Parts)

1. Lysis

General flow of NA Isolation (3 Parts):

• Opening of the cells, exposing the DNA or RNA to the reagents

• Break open cell membranes or protein capsids

• Chemical, physical and means are used to destroy the structure of these membranes

2. Removal of unneeded biomolecules

General flow of NA Isolation (3 Parts):

• Remove lipids, carbohydrates, proteins

• Some add enzymes to selectively remove DNA/RNA

  • If doing DNA isolation, we remove RNA

  • If doing RNA isolation, we remove DNA

3. Precipitation of Nucleic Acids

General flow of NA Isolation (3 Parts):

• Done by treatment with alcohol

• They are washed several times before dried and resuspended in water of buffer

Sample Preparation

Samples have to be prepped before starting the experiment:

• Ensure that the cells are exposed to lysis solution.

• Removing unwanted tissue.

• Weighing the proper amount of tissue.

• Pre-treating the cells if needed.

1. Suspension cells

2. Tissue samples

3. Microorganisms/Cultured animal cells

samples you can use in DNA preparation and extraction

Suspension Cells (e.g., Blood)

• Only nucleated cells are useful (e.g., white blood cells, not red blood cells).

• Cells are separated using centrifugation, differential gradient centrifugation, or differential lysis.

Differential gradient centrifugation (e.g., through Ficoll)

Sample Preparation:

• _______________________________________ separates cells based on density.

Differential lysis

Sample Preparation:

• ______________ using hypotonic solutions can selectively lyse unwanted cells while preserving target cells.

Tissue Samples

• Dissociate cells (l.e. homogenization in liquid nitrogen, using a tissue homogenizer, maceration, enzymatic separation)

Paraffin-embedded tissues;

xylene;

alcohol

Sample Preparation:

• __________________________ have to be deparaffinized using ______ and rehydrated using decreasing concentrations of _______

lysozyme;

Proteinase K;

mechanically lysed (using grinders, homogenizers, sonicators)

Sample Preparation: Microorganism/Cultured animal cells:

• Cells can be enzymatically lysed using enzymes like __________ or ___________ or can be _______________________________________

• detergent (1% sodium dodecyl sulphate)

• strong base (0.2M NaOH)

• Tris Base

• EDTA

• glucose

Sample Preparation: Microorganism/Cultured animal cells:

• needed treatment to break down bacterial cell walls.

8% sucrose, 8% Triton X-100 detergent, Tris buffer, and EDTA

Boiling in ________________________________________________ after lysozyme treatment releases DNA.

denatured DNA;

downstream processes

Microorganism sample preparation

• Procedures using strong bases and boiling result in ______________ which cannot be used for some applications like ____________

Organic Isolation Methods

DNA Extraction Methods:

• A combination of high salt, low pH, and an organic mixture of phenol and chloroform helps in the removal of lipids, carbohydrates and proteins.

Phenol-Chloroform

DNA Extraction Methods:

• dissolves hydrophobic contaminants (lipids, lipoproteins)

• collects cell debris

• removes most DNA-associated proteins.

RNase (Ribonuclease)

enzyme to remove RNA contaminants.

lower phase = organic phase; hydrophobic cells (lipids & membranes)

Addition of phenol and chloroform causes the formation of 2 phases upon centrifugation.

• lower phase = ______________________

upper phase = aqueous phase; hydrophilic molecules; DNA & RNA

Addition of phenol and chloroform causes the formation of 2 phases upon centrifugation.

• upper phase = ______________________

interphase = amphiphilic components; cell debris & proteins

Addition of phenol and chloroform causes the formation of 2 phases upon centrifugation.

• interphase = ___________________________

DNA Precipitation

Organic Isolation Methods:

• Add high concentration of salt (e.g., NaCl, KCl, ammonium acetate).

2:1 ethanol

1:1 isopropanol

Add ________ or _________ to precipitate DNA to the upper phase.

pellet

Organic Isolation Methods:

• Centrifuge to collect DNA as a _______.

70% ethanol

Organic Isolation Methods:

• Washing with ____________ removes excess salt.

TE buffer (10mM Tris, 1mM EDTA);

nuclease-free water.

DNA is then resuspended in ________________________ or _________________

Tris base

Maintains a stable pH (usually ~8.0), preventing DNA degradation.

EDTA

Binds divalent cations like Mg²⁺ and Ca²⁺. These ions are required by nucleases (DNases), so removing them prevents DNA breakdown.

TE Buffer

Best for long-term storage because it protects DNA from degradation.

Nuclease-free water

• Free from DNases and RNases.

• Does not contain EDTA, so it won’t interfere with downstream reactions like PCR (since PCR requires Mg²⁺).

• Best for immediate use, especially for PCR or enzymatic reactions.

Inorganic Isolation Methods

• '“salting out”

• Tries to remove the use of organic solvents and chemicals like phenol

• Safer and simpler for routine DNA extraction.

• Low pH and high salt conditions selectively precipitate proteins while leaving DNA in solution.

• DNA is then precipitated, washed and resuspended using similar ways asin organic extraction

Low pH;

high salt

Inorganic Isolation Methods:

• _______ and ________ conditions selectively precipitate proteins while leaving DNA in solution.

SDS

an ionic detergent which can break apart the cell by interfering with the interactions of membrane molecules, the phospholipids

Solid-Phase Extraction

• DNA binds to a solid matrix, usually silica, under certain conditions (often high salt).

• Other cellular components (proteins, lipids) do not bind and are washed away

silica column or beads

Solid-Phase Extraction:

• Lysate is applied to a _____________________ —- are usually small spin columns that fit inside microcentrifuge tubes.

viral and bacterial DNA

Solid-Phase Extraction:

• Used to isolate ________________ from serum, plasma, CSF, etc.

genetics;

molecular diagnostics;

oncology

Solid-Phase Extraction:

• Commonly used to isolate DNA from cells for use in _________, ___________ and __________ applications

string columns

term used for silica columns

silica spin column

SOLID-PHASE EXTRACTION:

• The cell lysate is placed in a ____________ which adsorbs/adheres DNA.

elusion

SOLID-PHASE EXTRACTION:

• last step

• separating DNA from the matrix so that it gets resuspended in the solution

• uses elusion solution

QIAGEN spin column

spin column for plasmid extraction

1. Crude lysis procedure

2. Proteolytic lysis of Fixed material

3. Extraction using chelating resin

4. Extraction through PCR

3 Other Isolation Methods:

Crude Lysis Procedures

4 Other Isolation Methods:

• Used when there are too many sample to process and you dont need clean DNA

• Having lysed cells releases enough DNA for the experiment

Proteolytic Lysis of Fixed Material

4 Other Isolation Methods:

• Example of crude lysis extraction

• Used for paraffin-embedded tissues.

1. decreasing concentrations of alcohols

2. proteases

Proteolytic Lysis of Fixed Material:

1. Rehydrate the tissue using ____________________

2. Use ____________ to lyse cells embedded in paraffin

Microdissection

Proteolytic Lysis of Fixed Material:

• _______________ is done to collect DNA straight from the tissues

• usses very small needle made of tungsten to cut the nucleus of the cell

• the nuclear components are then sucked up using an injection

Extraction using Chelating Resin

4 Other Isolation Methods:

1. addition of resin (chelex)

2. boiling (lysing)

3. centrifuge

Chelex

attracts everything to the bottom, except DNA

10% solution of resin (chelex) beads ;

boiling;

supernatant;

chelex and cell debris

Extraction using Chelating Resin:

• __________________________ is mixed with the specimen and then lysed through ______.

• Solution is centrifuged and DNA remains in the ____________.

• at the bottom, you will find the _________________

Extraction through PCR;

cells;

bacterial cells;

lysed;

4 Other Isolation Methods:

• makes use of _____ as the sample instead of DNA

• Instead of loading DNA into a PCR run, we would load __________ instead

• Microorganisms no longer have to be ______ beforehand

thermocycler

Extraction through PCR:

• Heat and buffers in the ____________ lyse cells and release DNA for amplification.

pick colony > replicate plate > add to SapphireAmp Fast PCR Master Mix (+ primers & H2O) > PCR > load 5 microliter directly to agarose gel wells > electrophoresis

steps in DNA extraction through PCR

PCR colony hybridization

4 Other Isolation Methods:

• used when you have a lot of sample to assay

• to save time and effort

• usually for determining if sample has plasmids

1. Selective Collection of Mitochondria

2. Total Genomic DNA Extraction

2 ways in isolating mitochondrial DNA

Selective Collection of Mitochondria

Isolating mitochondrial DNA:

• Enrich for mitochondria before extracting DNA.

• Methods:

  • Differential centrifugation – spins samples at different speeds to separate mitochondria from nuclei and other cell components.

  • Density gradient centrifugation – separates mitochondria based on their density.

• Once isolated, DNA is extracted directly from the mitochondria, giving mostly mtDNA.

Total Genomic DNA Extraction

Isolating mitochondrial DNA:

• Simpler approach: extract all DNA from the cell.

• The resulting DNA mixture contains:

  • Nuclear DNA

  • Mitochondrial DNA (present in high copy numbers)

• mtDNA can then be analyzed from the genomic DNA extract, often using PCR or sequencing methods that target mtDNA specifically.

RNAse

EXTRACTION OF TOTAL RNA

Before extracting RNA, _________ must first be eliminated

ice or 4°C

EXTRACTION OF TOTAL RNA

RNA extraction should mostly be done on ______ tempeature

T

RNA are way more easy to degrade than DNA

4 Ways to eliminate RNases

1. RNase eliminating sprays (e.g. RNase Away, RNase Zap)

2. Treament with DEPC (Diethyl Pyrocarbonate)

3. Baking

4. Nuclease-free disposable supplies are preferred

1. RNase eliminating sprays (e.g. RNase Away, RNase Zap)

4 Ways to eliminate RNases

• For the area, glassware sometimes, for the equipment

Treament with DEPC (Diethyl Pyrocarbonate)

4 Ways to eliminate RNases

• We do this with the reagents  We’ll also do this when rinsing glassware

Baking;

200 C;

300 C for three (3) to five (5) hours

4 Ways to eliminate RNases

• If you have glassware, you have to bake them usually this at temperatures of above ___ for very long amount of time

• so the one being used is ___________________________

Nuclease-free disposable supplies

4 Ways to eliminate RNases

• There are commercially available nuclease – free disposable supplies

• However, if you are a laboratory that does not have a lot of money, it is expensive to do RNA work with disposable all the time

• RNAses

• Sweat, tears, any other fluids

• ________ can come from many sources in the body

• ______________, etc., there’s a lot of sources and so, we always wear gloves that are fit and then we would always wear a mask as well

Lysis

EXTRACTION OF TOTAL RNA

____ is the first step in RNA extraction – just like DNA extraction (opens up the cell to expose RNA molecules)

osmosis ;

centrifugation

EXTRACTION OF TOTAL RNA

Reticulocytes in blood and bone marrow samples are lysed by ______ or

separated from WBCs (leukocytes) by ________.

liquid nitrogen;

buffer

EXTRACTION OF TOTAL RNA
Sample can be frozen in ______ or

kept in a ______ that inactivates RNases.

chemical or mechanical lysis.

EXTRACTION OF TOTAL RNA

Microbes are lysed using ______________

serum;

no need

EXTRACTION OF TOTAL RNA

Viral RNA can be directly taken from _______ or other biological samples.

There is ________ to separate viral RNA

Pathogenic microbes

EXTRACTION OF TOTAL RNA

___________ are best isolated from host cells before extraction.

detergent;

high salt (0.2–0.5 M NaCl);

RNAse inhibitors

LYSIS FOR EXTRACTION OF RNA

The cell lysis step for RNA isolation is done in _______ or phenol in the presence of _________ or _______

Guanidine thiocyanate

LYSIS FOR EXTRACTION OF RNA
___________ is a strong denaturant of RNAses and can be used instead of high salt buffers.

and RNase inhibitor

2-mercaptoethanol

LYSIS FOR EXTRACTION OF RNA

Strong reducing agents such as ____________ can also be added during this step.

phenol;

chloroform;

isoamyl alcohol

EXTRACTION OF RNA

After lysis

• proteins are extracted by —→

• proteins are denaturated by and promotes phase separation —→

• prevents foaming —→

acidic (ph 4–5).

EXTRACTION OF RNA

The organic phase must be ________

DNase

EXTRACTION OF RNA

_____ may be added at the lysis step or directly to extracted RNA to eliminate contaminating DNA.

This is you will destroy DNA fragments so you don’t contaminate your RNA

Centrifugation;

upper aqueous layer

EXTRACTION OF RNA

_________ facilitates phase separation and the __________ contains the RNA.

microcentrifuge tube;

1:1 isopropanol or 2:1 ethanol;

EXTRACTION OF RNA

The upper aqueous layer is transferred into a _______and then precipitated with ____

Centrifugation;

70-75% ethanol

EXTRACTION OF RNA

_________ pellets the RNA and the pellet is subsequently washed with _______ to remove any other impurities

nuclease-free water;

buffer (usually DEPC-TE)

EXTRACTION OF RNA

The RNA is then dried and resuspended in ________ or ________

acidification that pellets cell debris

which step in DNA extraction is absent in RNA extraction

Trizol

EXTRACTION OF RNA

a chemical that promotes cell lysis

already contains phenol

SOLID PHASE EXTRACTION OF RNA

80-90%

% of rRNA from the total RNA

2%

% of mRNA from the total RNA

EXTRACTION OF MRNA

columns

EXTRACTION OF MRNA

Around 80%–90% of total RNA is rRNA to extract mRNA which makes up roughly 2% of total RNA, the use of ________ is employed.

polyT or polyU

EXTRACTION OF MRNA

Column has _____ in order to attach to the tails, sometimes they also use _____ which also works

ologomers of thymine or uracil

EXTRACTION OF MRNA

To enrich mRNA yield especially if there are transcripts that are low copy numbers, etc., we have different protocols where would use_________ in the matrix so these are all on the column described earlier

polyA

EXTRACTION OF MRNA

The polyT or polyU oligomers will bind the ______ tail found exclusively on mRNA.

warmed, low-salt

EXTRACTION OF MRNA

After washing away residual RNA, polyA RNA is eluted by washing the column with _________

RT PCR/ Reverse Transcription PCR

MRNA EXTRACTION THROUGH PCR

This is a schematic for mRNA extraction protocol where we go straight to the __________

Step 1: MIx sample with lysis buffer

Step 2: Add biotin attached to polyDT or oligoDT, which would then attach to mRNA, then incubate

Step 3: Add 50 microleter of mix to Streptavidin-coated PCR tube (immobilization occurs)

Step 4: Wash 3 times with buffer

MRNA EXTRACTION THROUGH PCR

4 steps

Streptavidin

_____ has a very high affinity to biotin so the moment you have both, they would attach to each other

Since streptavidin is basically attached to the wall of the tube, mRNA also becomes attach to it