MOLBIO LAB - Nucleic Acid Extraction

Nucleic Acid Extraction

First step of any amplification experiment, no matter what kind of amplification method is used to detect a specific pathogen.

Nucleic Acid Extraction

Considered as a pre-analytical step in an amplification method.

1. Lysis
2. Precipitation
3. Binding
4. Washing
5. Elution

PRINCIPLES: (LPBWE)

Lysis

Nucleic acids are released and nucleases are denatured

Precipitation

brings the nucleic acid out of the solution

Binding

Nucleic acids to a membrane surface such as silicates.

- This binding is facilitated by the chaotropic salt conditions and high ionic strength of the lysis/binding

buffer.

Washing

Removal of unbound substances (e.g. proteins, cell debris, and PCR inhibitors) by several washing steps.

Elution

Purified nucleic acid from the membrane (the solution will now contain a purified nucleic acid)

elution buffer

The purpose of extraction is to release the nucleic acid from the cell to be used in subsequent procedures.

These target nucleic acid should ideally be free from contamination (e.g. proteins, carbohydrates, lipids, or other nucleic acids).

In the extraction method, there is the initial release of cellular material by breaking the cell and nuclear membrane called as cell lysis.

1958

Meselson and Stahl discovered the density gradient and centrifugation strategies

Demonstrates the semiconservative replication of DNA

Alkaline lysis procedure - 1-50kb of plasmid DNA

The problem with the alkaline lysis is that large chromosomal DNA proteins and proteins cannot renature properly when neutralized in acetate at low pH after alkaline treatment. In turn, it would form large aggregates instead, which precipitates out of the solution.

Meselson and Stahl

_________________discovered the density gradient and centrifugation strategies

Cesium Chloride/Ethidium Bromide Density Gradient Centrifugation

Used in the 1950s for DNA extraction

Principle: Use difference in density between cesium ion and water and intercalation of ethidium bromide which shows good results for separation of various DNase and the procurement of high-yield DNA.
CESIUM CHLORIDE/ETHIDIUM BROMIDE DENSITY GRADIENT CENTRIFUGATION

Limitations: Requires an expensive ultracentrifuge and considerable time, difficult to perform, ethidium bromide is harmful

Pre-treated

differential density gradient centrifugation or differential lysis

Ficoll

highly branched sucrose polymer; does not penetrate biological membranes

EDTA

protects DNA from damages by DNases (it inhibits the enzyme activity of DNases)

Inorganic Isolation Methods

"SALTING OUT"

Solid-Phase Isolation

-Introduced by McCormick et al. in 1989
-Involves insoluble siliceous core particle which functions similar to
phenol.

Magnetic Bead Method

- Modification of solid-phase extraction or isolation.

- Advantage: No need for repeated centrifugation, vacuum filtration,

column separation for washing and elution, and organic solvents

- Simple and convenient (only uses magnetic bead beads).

- This method is commonly used in automated extraction methods

such as miniMag (bioMerieux) and MagNA Pure (Roche).

- The beads have negative surface charge, and binds proteins and

cellular debris selectively.

- Extraction principles using magnetic silica particles

Anion Exchange Methods

Solid-phase anion-exchange chromatography
It is based on the interaction between negatively-charged
phosphates of the nucleic acid and positively charged surface
molecules of the substrate.
DNA binds to substrate under low salt conditions.
RNA, cellular proteins, metabolites, and other interferences-
washed away by medium-salt buffers
Pure DNA- eluted using high-salt buffer and recovered by alcohol
precipitation
DNA yield purity and its biological activity is equal to at least two
rounds of purification in the cesium chloride (CsCl) gradients, but
in much lesser time
Isolated DNA- size can be up to 150 kb

Filter Paper-Based Methods

- Store dried biological specimen and isolate

- The filter paper also contains compounds that kill microorganism

and inhibitory non-microbial degradation of DNA.

- Untreated: Fixed with methanol and DNA eluted

- Treated: Contains compounds that lyse biological samples and

bind nucleic acid

- Adequate for banking of DNA in dried blood spot- at least 19

months at ambient temperature

Crude Lysis

Large amounts of samples
Isolation of DNA from limited amounts
Isolation of DNA from challenging samples (e.g. fixed paraffin-
embedded tissues) o Involves simple lysis- yields sufficiently useful
DNA for amplification procedures
Proteolytic lysis of fixed material

1. Centrifugation- after the cell preparation are homogenized by

grinding on ice, the homogenate is then centrifuged.

2. Isolate total DNA (same procedure above)

ISOLATION OF MITOCHONDRIAL DNA:

2 approaches:

Diethyl pyrocarbonate (DEPC)

converts primary and secondary amines to carbamic acid esters

Diethyl pyrocarbonate (DEPC)

is added to water and buffers, except for Tris buffers

can cross-link RNAse proteins through intermolecular
covalent bonds, which render its insoluble

Vanandyl-ribonucleoside complexes

binds active sites of RNase enzymes

Macaloid clays

absorbs RNase proteins

RNA (80-90%)

consists of 2 components, large and small, which are visualized by agarose gel electrophoresis

mRNA (2.5-5%)

has a faint background underlying the ribosomal RNA

Reticulocytes

extracted using osmosis or centrifugation

Tissue

- kept frozen in liquid nitrogen or immersed in buffer

buffer

The ________ in the tissue inactivates intracellular RNases which is true for pancreas which contains large amounts of innate
RNases.

Bacterial and fungal RNA

used as chemical lysis or grinding in liquid nitrogen

Viral RNA

isolated directly from serum or other cell-free fluids (through spin-columns or beads)

Cell lysis

involves detergent or phenol in the presence of high salt
(0.2-0.5 M NaCl) or RNase inhibitors (Guanidine thiocyanate or 2-mercaptoehanol)

Extracted with phenol, in combination with chloroform and isoamyl
alcohol

 Acid phenol:Chloroform:Isoamyl alcohol in the ratio of 25:24:1

 Chloroform- enhances extraction of nucleic acid by denaturing

proteins and

 promoting phase separation

 Isoamyl alcohol- prevents foaming

(pH 4-5)

Organic phase should be acidic

DNase

added at the lysis step to produce an RNase-free DNase

Organic Extraction of Total RNA

The RNA is in the upper aqueous phase, and is then precipitated by addition of 2 volumes of ethanol or 1 volume of isopropanol.

Glycogen or yeast transfer RNA may be added at this step as a carrier to aid RNA pellet formation.

RNA is then precipitated and washed in 70% ethanol and resuspended in RNase-free buffer or water.

Solid-Phase Separation

Strong denaturing buffer conditions must be adjusted before application of the lysate to the column. The lysate is applied to the column in high salt chaotropic buffer.

The adsorbed RNA is washed with supplied buffers, and the DNase can be added directly to the

adsorbed RNA on the column to remove contaminating DNA.

It is the eluted and transferred to a new microcentrifuge tube, which

has the purified RNA.

• 1,000,000 eukaryotic cells or 10-50 mg of tissue= yields

about 10 μg of RNA

Isolatiom of Poly 4 (messenger) RNA

Uses oligomers of thymine or uracil.
Oligomers (termed as polyT or polyU) are immobilized on a matrix
resin column or beads. They bind the
polyA tail found exclusively on mRNA.
polyA RNA is eluted by washing the column with warmed, low-salt
buffer-containing detergent
1 μg of total RNA= yields 30-40 ng of mRNA Limitations:
Secondary structure competes with binding to the capture oligomer (the binding of polyA and polyU)
o The secondary structure are intra-strand or inter-strand
hydrogen bond.
mRNA with short polyA tails- does not bind efficiently
AT-rich DNA fragments might also bind
rRNA might co-purify with polyA RNA

Manual Methods

-Commercial kits (commercially available)

-Requires cost, time, demands, and labor intensity

Advantage: Uses non-corrosive agents

-Limitations:

-Reproducibility (since it is manual)

-Use of Ethanol

-High complexity test (according to CLIA’88)

Automated

-Easily divided by workload capacity

-Each laboratory can select an appropriate instrument based on

throughput

-Fast TAT

-Avoids pipetting errors (since machine is used)

-Constant reproducibility

-Diminishes cross-contamination (involved reduced handling steps)

-QC monitoring (since it is an automated instrument)

Limitations:

-Economic aspect- an expensive instrument and extraction reagents including disposables are needed

-Troubleshooting

Economic aspect- an expensive instrument and extraction reagents including disposables are needed

Troubleshooting

Personal Protective Equipment (PPE)

● When performing nucleic acid extraction, proper PPE must be worn inside the laboratory.

→ Gloves (2layers-innerandoutergloves)

→ Eyegoggles

→ N95 respirator

→ Disposable lab gown (solid front)

● Biosafety Cabinet (Class II BSC)
● Centrifuge
● Vortex
● Mini-Centrifuge
● Additional Equipments
● Safety Information

Equipments:

Biosafety Cabinet (Class II BSC)

→ Placed in the nucleic acid extraction area or sample preparation
area.
→ Before using it, the MT should check for its function, and decontaminate the BSC.

Centrifuge

→ Speed should be capable of 10,000 x g.
→ Must have a removable rotor placed inside the BSC
→ Must have a sealing biocontainment lid/cover inside.
→ It should push materials through the filter.

Vortex

→ Thoroughly mix samples and reagent
→ Variables peed, used inside the BSC

Mini-Centrifuge

→ Quickly spin MCTS (remove droplets from the lid)
→ Used inside BSC

Additional Equipments

Timer
Pipettes
Tuberackforthesamples
MicrocentrifugeTuberack Biohazardwastecontainers(forsolidandliquidwaste) Papertowels
Cold block

Safety Information

→ PPEmustbewornatalltimes.
→ Buffer NVL contains chaotropes- may form highly reactive compounds with bleach
→ Sample containers should only be opened inside the BSC to avoid contamination

Preparation of Extraction Reagents

● When opening a new kit for RNA extraction, the buffer and the carrier RNA should be prepared.

Buffer Preparation

● Prepare RB1, RBW, and RNW
● Supplied as a concentrate.
● Add appropriate amount of ethanol (96-100% concentration)
● Mark the cap of the bottle with the date that it was added with ethanol
and the initials of the one who added the ethanol to the buffer.
The alcohol that should be used is not denatured ethanol but absolute ethanol (96-100%).
● Check Buffer NVL- contains chaotropes which can form highly reactive compounds when combined with bleach
→ Itisimportantthatthebleachisnotaddeddirectlytothesample preparation waste.

Carrier RNA Preparation

● Prepare 1 μg/μL solution- by adding 370 μL of nuclease free water (rehydrated)
→ ThecarrierRNAisalyophilizedreagentfoundinthekit.
● Aliquot into sterile 1.5 mL MCTs, and stored at -20°C.
● Rehydrated RNA should be kept cold during use.
● Carrier RNA is added to improve the binding capacity of the mini-spin column when viral nucleic acids included in the sample are low copy, and it also protects the target nucleic acids from the chance of degradation due to RNase or residual RNase activity.

● (Magnetic Bead Method)
● Lysis
● Washing
● Elution

AUTOMATED RNA EXTRACTION

(Magnetic Bead Method)

Genolution NX-48S

Lysis

the nucleic acid binds to the magnetic beads.

Washing

wash the bead with the washing buffer

Elution

the nucleic acid is released from the magnetic beads, and the
magnetic beads are removed by a magnet.