M3: NUCLEIC ACID ISOLATION Flashcards by ERL JOFERSON CABAUATAN

Breaking of cells basically to liberate nucleic acid using physical and chemical means such as grinding or vortexing, and putting into a solution containing salt

Cell lysis

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Often a ____ is added to degrade nucleic acid-associated proteins and other cellular proteins in the removal of cellular debris

protease

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Alternatively, some of the cellular debris can be removed by ___

filtering the sample

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___ allows the isolation of the nucleic acid by changing its surface characteristics to clump nucleic acid materials together, and to make the structure more stable in solutions

Precipitation

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____ involves removing unwanted molecules from your nucleic acid material

Purification

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After the basic steps in nucleic acid extraction, the nucleic acid material is resuspended and stored at ___ for further downstream processes

-70C

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what is the purpose of nucleic acid extraction ?

release the nucleic acid content from the cell to isolate and purify using extraction methods

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Nucleases are very stable enzymes that catalyze the digestion of nucleic acids by ___

cleaving the phosphodiester bonds between nucleotides

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Nucleases, DNases and RNases are commonly found on ____

human hands
bodily fluids
contaminated water and reagents
laboratory instruments and surfaces
and  in the sample itself

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Maintaining a ___ in molecular diagnostics lab is important to ensure the integrity of nucleic acid being tested

nuclease-free environment

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What are the precautions that should be undertaken when extracting nucleic acids: wearing PPE ?

protect yourself from the sample but also to prevent contaminating the sample

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What are the precautions that should be undertaken when extracting nucleic acids: designated work areas with controlled airflow ?

solely for RNA and DNA extraction to reduce nuclease contamination

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What are the precautions that should be undertaken when extracting nucleic acids: Decontamination of lab surfaces, equipment and instruments are done with ?

nuclease inhibitors
10% bleach
70% ethanol

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What are the precautions that should be undertaken when extracting nucleic acids: Treat polypropylene tubes and tips with ___ to inactivate RNases

dimethyl pyrocarbonate (DMPC)
diethyl pyrocarbonate (DEPC)

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What are the precautions that should be undertaken when extracting nucleic acids: minimizing the number of freeze-thaw cycles ?

freezing and thawing changes the pH of the sample, leading to nucleic acid degradation

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In sample preparation what are among the issues to consider when choosing a particular technique ?

technical requirements
time efficiency
cost effectiveness

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In sample preparation these affect the success of molecular diagnostic applications

quality and quantity of recovered DNA

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Purifying DNA includes ___ (2) from prokaryotic or eukaryotic sources

isolation of plasmids

isolation of chromosomal or genomic DNA

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Under sample preparation, successful nucleic acid purification requires ___ (4)

effective disruption of cells or tissue
denaturation of nucleoproteins
inactivation of nucleases
removal of contaminants such as proteins, carbohydrates, lipids

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This liquid phase extraction method is based on the differential solubility of nucleic acid and other cellular components

Organic extraction

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An organic solution containing ___ and ___ is mixed with a cell lysate, and two phase liquid are separated

phenol

chloroform

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What components are extracted to the lower organic phase in organic solution ?

proteins
lipids
lipoproteins
hydrophobic contaminants

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What components are extracted to the upper aqueous phase ?

nucleic acid

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How is the nucleic acid from the aqueous phase in organic extraction recovered ?

alcohol precipitation

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In the phenol-chloroform method this is used in the liberation of DNA from a cell

sodium dodecyl sulfate

What is the main reagent for phenol-chloroform method ?

phenol-chloroform-isoamylalcohol

In the phenol chloroform method of organic extraction this prevents emulsification which promoted precipitation of DNA

Isoamyl alcohol

Proteinase K is a serine protease from the fungus ___

Engyodontium album

This form of organic extraction is appropriate for whole eukaryotic or bacterial cells

Phenol-chloroform method

At what pH is organic DNA extraction formed at ?

pH 7-8

At what pH is organic RNA extraction formed at ?

pH 4-6

Pretreatment of this in DNA extraction can efficiently separate DNA from polysaccharide contamination

cetyltrimethyl ammonium bromide

How is the acidic pH achieved in RNA extraction ?

Detergent or phenol in the presence of high salt (0.2-0.5 M NaCl) RNAse inhibitors

This is a strong denaturant of RNAses and can be used instead of high-salt buffers Strong reducing agents such as ____ may also be added during RNA extraction

Guanidine isothiocyanate (GITC) 2-mercaptoethanol

This organic extraction method is used for impure DNA extracts or samples with complex chemical matrix

Cetyltrimethylammonium Bromide (CTAB)

The inorganic extraction leaves the DNA in the solutions by using high concentrations of salts such as ___ (3) to selectively precipitate proteins

ammonium acetate potassium acetate sodium chloride

This solid phase extraction method employs selective binding of nucleic acid to a solid matrix in a column as a cell lysate is applied to the column

Column-based extraction

In column-based extraction the first wash buffer is passed to remove ___ and a second one is passed to wash off ___

proteins salt contaminants

In column-based extraction an appropriate ___ or ___ is applied to the column to release the nucleic acid from the matrix into a microcentrifuge tube

elution PCR-grade water

What is the principle of silica extraction ?

selective binding of the negatively charged DNA with the silica surface covered with positively charged ions

Contaminants can be washed out and extracted DNA is eluted from the silica particles using ___ or ___

distilled water a buffer such as Tris-EDTA

This solid phase extraction method utilizes small particles with paramagnetic nanoparticles coated with DNA-binding antibody or polymer with specific affinity to DNA

Magnetic bead-based extraction

In magnetic bead-based extraction magnetic pellets can be eluted via the ___ method

ethanol precipitation

This DNA extraction method principle is based on DNA molecules and other simple and complex molecules in a complex matrix can be separated based on net molecular charge or molecular size Used for crude cell lysates

Chromatography-based extraction method (Ion Exchange Chromatography and Molecular Exclusion Chromatography

In the EtBr-CsCl gradient centrifugation method what is the centrifugation program ?

10,000 - 12,000 rpm for >10 hours

What is the purpose of cesium chloride in the EtBr-CsCl gradient centrifugation method ? (2)

allows interaction with negatively charged DNA Cesium is a heavy metal, it helps in the isolation of DNA via density alone

What is the purpose of EtBr in EtBr-CsCl gradient centrifugation method ?

acts as an intercalating agent and allows supercoiled DNA to accumulate at lower densities

From the other DNA extraction methods this is used for plasmid-containing samples

EtBr-CsCl gradient centrifugation method

This DNA extraction method principle is where the sample is suspended in an alkaline solution containing NaOH and SDS detergent, causing the denaturation of chromosomal DNA

Alkaline extraction

In alkaline extraction ___ is then added to neutralize the solution, causing the chromosomal DNA to renature and precipitate from the solution

Potassium acetate

From the other DNA extraction methods this is used for the extraction of plasmid DNA

Alkaline extraction

Cellulose-based paper is impregnated with ___, ___, and ___ that facilitate the extraction of DNA

buffers detergents chelating agents

In chelex-100 extractions Chelex resin is a ___ material which ____

styrenedivinylbenzene chelates metal ions that act as cofactors for DNAses

What is the use of chelex in Chelex-100 extraction ?

Chelex prevents DNA from being digested by DNAses during boiling, which is done to denature proteins and DNA

From the other DNA extraction methods this can be used for tissue samples

Chelex-100 Extraction

This DNA extraction method principle has a spin plate composed of a 96-well plate with a hole drilled into the bottom of each well containing a disc of Whatman filter paper

Filter paper-based DNA extraction method

From the other DNA extraction methods this can be used for plant cell samples

Filter paper-based DNA extraction method

CTAB is useful for purification of nucleic acid from organisms which produce large quantities of ____ such as ___ and certain ___

polysaccharides plants gram-negative bacteria

What are the disadvantages of CsCl-EtBr method ?

complicated expensive time-consuming large scale bacterial cultures

In the nucleic acid extraction of bacteria and fungi cell walls can be lysed by ___ and ___ and alternatively by ____

high pH and detergents broken mechanically by grinding or by vigorously mixing with glass beads

Nucleic acid in human blood or bone marrow comes mostly from ___

white blood cells (WBCs)

___ added to the blood or bone marrow aspirate collected sample will prevent clotting, which will trap the WBCs

Anticoagulants

For differential density-gradient centrifugation, whole blood or bone marrow mixed with isotonic saline is overlaid with ___

Ficoll

In nucleated cell suspension of blood and bone marrow aspirates ___ is a highly branched sucrose polymerthat does not penetrate biologicalmembranes

Ficoll

Upon centrifugation of blood and bone marrow aspirates, the mononuclear WBCs settle in the Ficoll gradient that is below the ____ and above the ____

less dense plasma components polymorphonuclear cells and RBCs

Another method in blood and bone marrow aspirates cell suspension used to isolate nucleated cells takes advantage of the differential osmotic fragility wherein

Incubation of whole blood or bone marrow in hypotonic buffer or water will result in the lysis of the RBCs before the WBCs

For plasma with the high sensitivity achievable from amplification procedures, these sources of circulating nucleic acids can be detected for purposes of diagnostic and prognostic analyses. Such a test is called a ____

liquid biopsy

Fresh or frozen tissue samples are dissociated before DNA isolation procedures can be started like ___ (3)

Grinding the frozen tissue in liquid nitrogen Homogenizing the tissue Mincing the tissue using a scalpel

___ is the least damaging among tested fixatives, with ___, such as Bouin’s and B-5, being the worst for DNA recovery

Buffered formalin mercury based fixatives

For RNA isolation specimen collection consideration after cleaning, glassware must be baked for __ hours at __°C to inactivate the RNases.

4-6 hours 400°C

___ can be added to water and buffers (except for Tris buffer) to inactivate RNases permanently

Diethyl pyro carbonate (DEPC)

____ protects the DNA from damage by DNase present in the environment of DNA preparations intended for long-term storage

EDTA

For solid phase isolation ___ can be used to specifically select for plasmid DNA because chromosomal DNA will not renature properly upon neutralization and form a precipitate

Alkaline lysis

____ is applied with the DNA to enhance recovery, preventing irreversible binding of the small amount of target DNA

Carrier RNA or DNA

Grinding in alkaline buffers with reducing agents such as ___ will protect the mitochondria during the isolation process

β-mercaptoethanol

When dissociating tissue, the sample should be kept frozen in ___ that will inactivate intracellular RNases

liquid nitrogen or immersed in buffer

This provides a rough estimation of the purity of a nucleic acid samples whether its DNA or RNA

A260/A280 ratio

A reduction in the A260/A280 ratio detects ____ contamination

protein

An increase in the A260/A280 ratio detects ____ contamination

RNA

In a buffered solution, pure dsDNA has an A260/A280 ratio of ___

1.85-1.88

In a buffered solution, pure RNA has an A260/A280 ratio of ___

2.1-2.2

How do you discriminate whether your sample is RNA or DNA ?

If intended sample is: RNA - treat sample with RNAse-free DNAse to remove contamination from DNA DNA - treat sample with DNAse-free RNAse

The ___ ratio is an indicator of contamination of the nucleic acid sample by other non-protein molecules

A260/A230

There are several molecules which have high absorbance at 230 nm such as ___ (6)

``` guanidine thiocyanate (GTC) guanidine hydrochloride (GuHCl) EDTA ``` Non-ionic detergents like: Triton X-100 Tween20 phenol

In common laboratory practice, DNA and RNA samples with A260/A280 and A260/A230 of ___ are considered suitable for use in most downstream applications such as polymerase chain reaction and gel electrophoresis

>1.8

What are the target molecules for PicoGreen ?

dsDNA

What are the target molecules for Hoescht H33258 ?

dsDNA

What are the target molecules for Ethidium bromide ?

dsDNA, RNA

What are the target molecules for RiboGreen ?

RNA

What are the target molecules for Oligreen ?

ssDNA, oligo-DNA

what is the standard coefficient of dsDNA ?

50 ug/mL

What is the standard coefficient of ssRNA ?

40 ug/mL

what is the standard coefficient of ssDNA ?

33 ug/mL

What dye has a measurement range of 0.1-10 ug/mL ?

Hoescht H33258 | Ethidium bromide

What dye has a measurement range of 1-1000 ng/mL ?

RiboGreen

What dye has a measurement range of 0.1-1000 ng/mL ?

Oligreen

What dye has a measurement range of 0.025-1000 ng/mL ?

PicoGreen

This is due to the fact that some of the energy of the excited fluorophore is lost through molecular vibrations that occur during the brief lifetime of the molecules excited state.

Stokes shift

Which migrates faster through the pores in gel medium, smaller or larger nucleic acids ?

smaller nucleic acids

What may happen to the nucleic acid in gel electrophoresis if the running time is too long ?

smaller nucleic acid fragments run off the gel

A commonly used fluorescence stain is used in the electrophoresis method

Ethidium bromide

___ was introduced as an alternative fluorescence stain which has a lower toxicity and higher sensitivity

SYBR green

What are some of the instances explaining a smeared DNA band in gel electrophoresis ? (3)

protein contamination nuclease activity too much salt in the sample

M3: NUCLEIC ACID ISOLATION Flashcards

(103 cards)