MOLBIO - MT122 - Laboratory 6 - Nucleic Acid Extraction Part 1

Nucleic Acid Extraction

- First step of any amplification experiment, no matter what kind of amplification is used to detect a specific pathogen
- Is considered as a pre-analytical step in an amplification method

Precipitation

Principle of Nucleic Acid Extraction

- Brings the nucleic acid out of the solution

Lysis of Sample Material/ Lysis

Principle of Nucleic Acid Extraction

- Nucleic acids are released and nucleases are denatured

Washing

Principle of Nucleic Acid Extraction

- Removal of unbound substances like proteins, cell debris, and PCR inhibitors

Elution of Purified Nucleic Acid Extraction

Principle of Nucleic Acid Extraction

- The last step in nucleic acid extraction
- Will now contain the purified nucleic acid

Binding of Nucleic acids to a membrane surface such as silicates/ silica

Principle of Nucleic Acid Extraction

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

Isolation of DNA

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

Meselson and Stahl

They discovered the density gradient and centrifugation strategies
to demonstrate the semiconservative replication of DNA

True

True or False: In 1958, Meselson and Stahl found out the differences in solubility of large chromosomal DNA is more than 50 kbp plasmids and proteins in alkaline buffers

True

True or False: The problem with alkaline lysis is that large chromosal DNA 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

Alkaline lysis procedure

1 to 50 kb of plasmid DNA

Cesium Chloride/ Ethidium Bromide Density Gradient Centrifugation

First nucleic acid extraction introduced in the 1950s

Cesium Chloride/ Ethidium Bromide Density Gradient Centrifugation

This requires an expensive ultracentrifuge and considerable time, difficult to perform, and harmful

20-50 microgram

Preparation of the Sample

What is the expected yield of blood?

- Serum, plasma, CSF
- Dried Blood
- Saliva
- Buccal cells
- Bone, Teeth
- Hair Follicles
- Fixed Tissues
- Feces

Specimens adequate for analysis with DNA amplification

III. Buccal cells

Which of the following does not belong to the group?

Specimens adequate for analysis without DNA amplification

I. Blood
II. Bone Marrow
III. Buccal cells
IV. Solid tissue
V. Cultured cells

Ficoll

Highly branched surcrose polymer; does not penetrate biological membranes

True

True or False: Mononuclear WBCs settle into layers in the Ficoll gradient. Below are less dense plasma components while the above contain PMNs and RBCs

WBCs- pelleted by centrifugation
RBCs- membranes (ghosts) and Hgb in suspension and solution

Differences in osmotic fragility of RBCs and WBCs

WBCs - ______________________
RBCs - _____________________________________

Fresh or Frozen tissue

When using this sample, it should be dissociated first before DNA isolation procedure would take place

Fixed embedded tissue

This tissue shoud be deparaffinized

Xylene

Fixed embedded tissue is deparaffinized by soaking in?

Xylene

3 isomers of dimethyl benzene

Decreasing concentration of ethanol

Fixed embedded tissue is rehydrated in __________________________________________

Lysozyme or Zymolyase

Tough cell walls are digested by using ________________

Lysozyme or Zymolyase

It digests cell wall polymers and are commercially available

True

True or False: Microorganisms are treated with detergent such as 1% sodium dodecyl sulfate and strong base (0.2M NaOH) in the presence of Tris base, EDTA, and glucose

Sucrose, 8%

Microorganisms

Boiling: 8% ________, _____ Triton X-100 detergent, Tris Buffer, and EDTA. This result releases DNA immediately precipitated with alcohol

Phenol and Chloroform

- Resulting extraction is biphasic emulsion forms
- Dissolves hydrophobic contaminants such as lipids and lipoproteins

Cetyltrimethylammonium bromide

A cationic detergent that separates DNA from polysaccharide contamination

III, I, IV,II

Arrange the Following:

General Schemes of Organic DNA Isolation

I. The upper phase contains DNA and is collected and precipitated using ethanol or isopropanol in a high concentration of salt
II. DNA pellet is dissolved in rehydration buffer such as 10 mM of Tris and 1 mM of EDTA or water
III. The phenol and chloroform are added, which forms a biphasic emulsion. After centrifugation, a hydrophobic layer will settle on the bottom which contains lipids and other contaminants
IV. The DNA precipitate is then collected by centrifugation, and excess salt is removed by rinsing the pellet in 70% ethanol, then subjected to centrifuge and discard ethanol supernatant

False

Ethanol- Denatured
Isopropanol- Undenatured/pure

True or False:

Ethanol- Undenatured/pure
Isopropanol- Denatured

EDTA

- Protects DNA from damages by DNAses
- Useful in restriction enzyme digestion or PCR

- 10 mM Tris
- 1 mM of EDTA

Give the component of the Tris-EDTA buffer

Inorganic Isolation Methods

Also known as Salting out

Inorganic Isolation Methods

- It involves the removal of proteins and other contaminants may be inefficient
- Did not provide efficient recover of clean DNA
- Uses low pH and high salt conditions

Isopropanol

Inorganic Isolation Methods

DNA is precipitaed using _________ and now in a form of a pellet

Purity-variable

For Inorganic isolation methods, DNA yielded is _____________

Solid-Phase Extraction

Insoluble siliceous core particle which functions similar to phenol

Solid-Phase Extraction

- Introduced by McCormick et al in 1989
- Safer and cross contamination is reduced
- Rapid and comparably effective

True

True or False: According to Meijer et al: Phenol/chloroform method cause DNA loss

- Silica matrices
- Glass particles
- Diatomaceous earth
- Anion-exchange carrier

Siliceous core particles are replaced by?

Diatomaceous earth

Source of silica particles in solid-phase isolation

20-50 kilobase

The average size of genomic DNA isolated using Silica Gel membrane technique is ___________ of genomic DNA

20-50 kilobase of genomic DNA

Not suitable for high molecular weight genomic DNA because the size of HMW DNA is >100 kilobase

- Guanidine hydrochloride
- Guanidine isothiocyanate
- Sodium iodide
- Sodium Perchlorate

Selective absorption of nucleic acides to silica in the presence of high concentrations of chaotropic salts:

IV, I, V, II, II, VI, VIII, VII

Arrange the following:

Steps in Isolation of DNA on Solid-phase

I. Cell lystae if applied to a column in a high salt buffer
II. Then washed with buffer
III. The DNA adsorbs to the solid matrix in the spin columns
IV. Cell lysis and the release of nucleic acids
V. The DNA adsorbs to the solid matrix in the spin columns
VI. Then DNA is eluted in a specific volume of water/ tris EDTA or other low salt buffer
VII. Now we have a pure DNA
VIII. Once eluted, it is transferred to another microcentrifuge tube

Magnetic beads

Coated with silica particles allows isolation of genomic DNA w/o centrifugation or vacuum processing

Magnetic Bead Method

- Modification of solid-phase extraction
- This method is commonly used in automated extraction methods such as miniMag (bioMerieux) and MagNA Pure (Roche)
- Simple and convenient
- The beads have a negative surface charge and bind proteins and cellular debris selectively

IV, II, III,I

Arrang the following:

Magnetic Bead Method

I. The magnetic silica particle are separated from the eluate by the magnetic device
II. The magnetic device attracts all the magnetic silica — enabling the system to purify the nucleic acids through several washing steps
III. Heating step. This will relaeases the nucleic acids from the magnetic silica particles
IV. During incubation of the lyse sample all the target nucleic acids are captured by the magnetic silica particles / magnetic beads

Anion-Exchange methods

- DNA binds to substrate under low-salt conditions
- Solid-phase anion-exhange chromatography
- RNA, cellular CHONs, metabolites are washed away by medium-salt buffers

up to 150 kb

Anion-Exchange Method

Size of Isolated DNA = _____________

Filter paper-based methods

- Contains compounds that kill microorganisms and inhibitory nonmicrobial degradation of DNA
- Store dried biological specimen and isolate

19

Filter Paper-Based Methods:

Adequate for banking of DNA in Dried Blood Spot for at least _____ months at ambient temperature

True

True or False:
Filter Paper-Based Methods

If untreated- Fixed with methanol and DNA eluted
Treated- contains compounds that lyse biological samples and bind nucleic acids

Crude Lysis

- Useful for screening large number of samples, limited amount of DNA is available, challenging samples
- Involves simple lysis that yields sufficiently useful DNA for amplification procedure

Fixed Tissue

Uses thin sections for analysis

Paraffin-embedded specimen

Dewaxed with xylene and rehydrated before nucleic acid isolation

PCR (polymerase chain reaction)

Digested through tris buffer and Proteinase K

Proteinase K

digests proteins, lyses cells, inactivates other enzymes

Chelex

Cation-chelating resin, cells are lysed by boiling 10% chelex resin bead is mixed with specimen

Extraction with Chelating Resin

-Used for forensic applications
- Purification of DNA from clinical samples and fixed, paraffin-embedded specimens

Whatman

DNA extraction/ storage cards

Sigma or Epicentre Technologies

Rapid Lysis Method

Other Rapid Extraction Methods

Provides sufficiently clean DNA that can be used for amplification

- Centrifugation
- Isolate Total DNA

Give the two approaches for the isolation of Mitochondrial DNA

III. Mitochondrial DNA is analyzed within the total DNA background

Which of the following does not belong to the group?

Centrifugation

I. Centrifuged first at low speed (700-2600 x g)
II. Mitochondria is lysed with detergent and the lysate is treated with proteinase
III. Mitochondrial DNA is analyzed within the total DNA background
IV. It is precipitated with ethanol and resuspended in water

True

True or False: RNA is labile due to the ubiquitous RNases

True

True or False: When isolating RNA, it requires strict precautions to avoid sample RNA degradation

True

True or False: RNases renature even after autoclaving (and become active at a wide range of temperature even below -20°C) and even after heating

4-6 hours, 400 degrees celsius

When using reusable glasswares, after cleaning of the glasswares, it must be baked for ________ at _______ to inactivate RNases

Diethyl pyrocarbonate (DEPC)

- converts primary and secondary amines to carbamic acid esters
- can cross-link RNase proteins through intermolecular covalent bonds, which render its insoluble

False

except to Tris buffers

True or False: DEPC is added to water and buffers, especially Tris buffer

Vanandyl-ribonucleoside complexes

Binds active sites of RNase enzymes

Macaloid clays

It 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 a buffer
- The buffer 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)

25:24:1

Acid phenol:Chloroform:Isoamyl alcohol in the ratio of

Isoamyl alcohol

It prevents foaming

DNase

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

70% ethanol, RNase-free buffer or water

Organic Extraction of Total RNA

RNA is then precipitated and washed in _____________ and resuspended in __________________

Solid-Phase Preparation

- The lysate is applied to the column in a high salt chaotropic buffer
- It is eluted and transferred to a new microcentrifuge tube, which has the purified RNA
- 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

Yiels about 10 microgram of RNA

1,000,000 eukaryotic cells or 10-50 ug of tissue

Isolation of POLY A (Messenger RNA)

Uses oligomers of thymine or uracil

Oligomers

- Termed as polyT or polyU
- Immobilized on a matrix resin column or beads. They bind the polyA tail found exclusively on mRNA

polyA RNA

It is eluted by washing the column with warmed, low-salt buffer-containing detergent

30-40 ng of mRNA

1 μg of total RNA = yiels _____________

False

It does not bind efficiently

True or False: mRNA with short polyA tails does bind efficiently

True

True or False: rRNA might co-purify with polyA RNA

Manual Method

Requires cost, time, demands, and labor intensity
Uses non-corrosive agents

- Reproducibility
- Use of ethanol
- High complexity test

Give the Manual Method Limitations

b. Slow TAT

Which of the following is not true for Automated Method?
a. Easily divided by workload capacity
b. Slow TAT
c. Avoids pipetting errors
d. Constant reproducibility
e. All of the choices

c. High complexity test

All of the following are limitations of Automated Methods except:

a. Economic aspect
b. Trouble shooting
c. High complexity test
d. None of the above

High Pure Roche Applied Science

Nucleic acid capture by glass fiber fleece immobilized in a special plastic filter tube and subjected to centrifugation

QIAamp Qiagen

Nucleic acid capture by silica gel membrane placed in tube column and subjected to centrifugation or vacuum conditions