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 of Nucleic Acid Extraction (5)

Lysis

Identify Principle:

- Nucleic acids are released and nucleases are denatured

Precipitation

Identify Principle:

- Brings the nucleic acid out of the solution

Binding

Identify Principle:

- 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

Identify Principle:

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

Elution

Identify Principle:

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

Cell lysis

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

Density gradient and Centrifugation strategies

- Discovered by Meselson and Stahl in 1959

- Demonstrates the semiconservative replication of DNA

- In alkaline lysis procedure, only 1-50kb of plasmid DNA are allowed since if too large, it will form large aggregates

Cesium Chloride/Ethidium Bromide Density Gradient Centrifugation

- Used in the 1950s for DNA extraction

- PP: Uses the 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.

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

Nucleated cells in suspension

- Examples of this sample are blood and BM specimens

- These specimens should be PRE-TREATED

- WBCs extracted, then blood/BM specimens are then mixed with isotonic saline, then overlaid with Ficoll

Ficoll

- Highly branched sucrose polymer; does not penetrate biological membranes

- Where WBCs will settle

- Washed by at least 2 rounds of resuspension and centrifugation saline

Less dense plasma components ; PMNs and RBCs

Using Ficoll, what can be found BELOW? How about ABOVE?

Tissue samples

- This sample can be fresh or frozen

- Should be dissociated first before DNA isolation

Xylene ; ethanol

Fixed embedded tissue should be deparaffinized by soaking in ______ which is 3 isomers of dimethyl benzene.

Afterwards it is rehydrated in decreasing concentration of _______

Microrganisms

- These samples have tough cell walls, so they are digested by lysozyme or zymolase

- Alternatively, grinding or vigorously mixing with glass beads can be performed

- Can also be treated with detergent and strong base

Phenol and chloroform

In Organic Isolation Methods, this is in a biphasic emulsion

Fungi

In Organic Isolation Methods, these have small amounts of DNA

RNase

In Organic Isolation Methods, these are added to avoid RNA contamination

Organic Isolation Methods

- Phenol and chloroform are added

- The upper phase contains DNA and is collected and precipitated using ethanol or isopropanol in a high concentration of salt

- DNA pellet is dissolved in rehydration buffer

- Less volatile, precipitates DNA at room temperature, lesser volume is added for precipitation

Inorganic Isolation Methods

- Salting Out

- Initially cant provide clean DNA, so additional treatment needed ; meaning DNA yield and purity are variable

- Use low pH and high salt, then add Isopropanol to precipitate DNA in a pellet

Solid-Phase Isolation

-Introduced by McCormick et al. in 1989

-Involves insoluble siliceous core particle which functions similar to

phenol, and the addition of Cell lysate

- Safer and cross-contamination is reduced.

- Suitable for PCR and southern blot analysis

Diatomaceous earth

What is a source of Silica particles?

Magnetic Bead Method

- Modification of solid-phase extraction or isolation.

- No need for centrifugation, etc

- Commonly used in automated extraction methods

- Uses beads with negative charge which binds proteins and cellular debris

Anion Exchange Methods

- Solid-phase anion-exchange chromatography

- This is based on the interaction between negatively-charged phosphates of the nucleic acid and positively charged surface molecules of the substrate

-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

- Uses filter paper that can kill microorganisms and inhibit non-microbial degradation of DNA.

- Adequate for banking of DNA in dried blood spot: At least 19 months at ambient temperature

Crude Lysis

- For large amount of samples

- Isolation of DNA from limited amounts and challenging samples

-Involves simple lysis

Fixed tissue

In Crude Lysis, what sample should be in thin sections?

Paraffin-embedded specimen

In Crude Lysis, what sample should be dewaxed with xylene and rehydrated before nucleic acid isolation?

Simple screening methods

In Crude Lysis, what method uses cells lysed with detergents?

PCR

In Crude Lysis, what method uses Tris buffer and Proteinase K?

Proteinase K

This substance digests proteins lysing the cells and inactivate other enzymes.

Chelex / Cation-chelating resin

A Crude lysis substance that is only used in forensic applciations

Centrifugation ; Isolate total DNA

The 2 Methods in Isolation of DNA

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

- Converts primary and secondary amines to carbamic acid esters

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

In extraction of Total RNA:
- Extracted using osmosis or centrifugation

Tissue

In extraction of Total RNA:
- 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

In extraction of Total RNA:
- Used as chemical lysis or grinding in liquid nitrogen

Viral RNA

In extraction of Total RNA:
- Isolated directly from serum or other cell-free fluids (through spin-columns or beads)

Cell lysis

In extraction of Total RNA:
- Involves detergent or phenol in the presence of high salt
(0.2-0.5 M NaCl) or RNase inhibitors (Guanidine thiocyanate or 2-mercaptoehanol)

25:24:1

In extraction of Total RNA, Acid phenol:Chloroform:Isoamyl alcohol should be in what ratio

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, which is what range of pH?

DNase

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

Organic Extraction of Total RNA

- RNA precipitated by addition of 2 volumes of ethanol or 1 volume of isopropanol.

- Glycogen or Yeast transfer RNA can be added as a carrier

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

2 volumes ; 1 volume

What is the volume of ethanol if it is used in Organic Extraction of total RNA? How about isopropanol?

Solid-Phase Separation

- Uses a lysate to the column in high chaotropic buffer

-Strong denaturing buffer conditions must be adjusted before application of the lysate to the column.

-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

- 1 μg of total RNA = yields 30-40 ng of mRNA

- Secondary structure competes with binding, mRNA with short polyA tails- does not bind efficiently

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 (2 layers)

→ 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

→ PPE must be worn at all times.

→ 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

Carrier RNA Preparation

● Prepare 1 μg/μL solution- by adding 370 μL of nuclease free water (rehydrated)
→ The carrier RNA is a lyophilized reagent found in the kit.

● 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.

15-25C ; ethanol ; 56C

Before Starting:

1. Equilibrate samples to room temperature (______).

2. Check that buffer RB1, RBW, and RNW have has ________

added.

3. Check buffer NVL for precipitate, warm the bottle at _____ until

it is clear and the crystals are gone.

4. Thoroughly clean the BSC

True

True or False: When working inside the BSC, proper workflow should be followed (from
clean area→ dirty area).

Genolution NX-48S

AUTOMATED RNA EXTRACTION:

What equipment uses Magnetic Bead Method?

Lysis

AUTOMATED RNA EXTRACTION:

The nucleic acid binds to the magnetic beads.

Washing

AUTOMATED RNA EXTRACTION:

Wash the bead with the washing buffer

Elution

AUTOMATED RNA EXTRACTION:

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