M3: NUCLEIC ACID ISOLATION Flashcards
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
Often a ____ is added to degrade nucleic acid-associated proteins and other cellular proteins in the removal of cellular debris
protease
Alternatively, some of the cellular debris can be removed by ___
filtering the sample
___ 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
____ involves removing unwanted molecules from your nucleic acid material
Purification
After the basic steps in nucleic acid extraction, the nucleic acid material is resuspended and stored at ___ for further downstream processes
-70C
what is the purpose of nucleic acid extraction ?
release the nucleic acid content from the cell to isolate and purify using extraction methods
Nucleases are very stable enzymes that catalyze the digestion of nucleic acids by ___
cleaving the phosphodiester bonds between nucleotides
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
Maintaining a ___ in molecular diagnostics lab is important to ensure the integrity of nucleic acid being tested
nuclease-free environment
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
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
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
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)
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
In sample preparation what are among the issues to consider when choosing a particular technique ?
technical requirements
time efficiency
cost effectiveness
In sample preparation these affect the success of molecular diagnostic applications
quality and quantity of recovered DNA
Purifying DNA includes ___ (2) from prokaryotic or eukaryotic sources
isolation of plasmids
isolation of chromosomal or genomic DNA
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
This liquid phase extraction method is based on the differential solubility of nucleic acid and other cellular components
Organic extraction
An organic solution containing ___ and ___ is mixed with a cell lysate, and two phase liquid are separated
phenol
chloroform
What components are extracted to the lower organic phase in organic solution ?
proteins
lipids
lipoproteins
hydrophobic contaminants
What components are extracted to the upper aqueous phase ?
nucleic acid
How is the nucleic acid from the aqueous phase in organic extraction recovered ?
alcohol precipitation
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
