– NUCLEIC ACID ISOLATION AND TECHNIQUES IN EXTRACTION Flashcards
1
Q
extract nucleic acid/ DNA/ genetic composition of a microorganism
A
GOAL OF MOLECULAR BIOLOGY
2
Q
_____ - sample required: DNA
▪ Ex: Sars- COV 2 (RNA virus)
▪ RNA is converted to DNA
A
RT-PCR
3
Q
_______ first to isolate or extract DNA from human cells.
discovered it when he was studying the chemical nature of the nuclei of white blood cell (lymphocytes)
A
1869
fRIEDRICH MIESCHER
4
Q
_________ white substance released from the WBC
A
NUCLEIN
5
Q
Nucleic acid extraction can be divided into three steps:
A
- Breaking open of the tissues and cells
- Removing proteins, lipids, and other contaminants.
- Transferring nucleic acids to water or a buffer solution that will preserve them without interfering w/ subsequent work.
6
Q
________ process of transferring nucleic acids to a molecular biology grade water/ buffer
A
ELUTION
7
Q
________ = solution for processing
A
ELUATE
8
Q
Buffer solution - __________
A
Tris EDTA / A buffer
9
Q
Three steps (summary) of NUCLEIC ACID ISOLATION AND
TECHNIQUES IN EXTRACTION
A
o Lysis
o Separation
o Precipitation and resuspension
10
Q
Initial laboratory procedures for DNA isolation.
A
DENSITY GRADIENT CENTRIFUGATION STRATEGIES
11
Q
Procedure for separating particles (e.g., viruses, ribosomes, or DNA), in which the sample is placed on a pre-formed gradient, such as _______ or __________.
A
SUCROSE or CESIUM CHLORIDE
12
Q
Upon centrifugation, either by _______ or ___________, macromolecules are bonded in the gradient and can be collected as ___________.
A
- rate-zonal or equilibrium procedures
- PURE FRACTION
13
Q
_____________ – used such a method in 1958 to demonstrate SEMICONSERVATIVE REPLICATION OF DNA
A
- MESELSON AND STAHL
14
Q
THERE ARE TWO TYPES OF DENSITY GRADIENT
CENTRIFUGATION STRATEGIES:
A
1 DIFFERENTIAL PELLETING
2 DENSITY GRADIENT CENTRIFUGATION
a Rate-Zonal Centrifugation
b Isopycnic Centrifugation
15
Q
o lower density gradient is used and cells are principally separated on size differences (gradient will float).
o Uses lower density gradient which cause the gradient to float over DNA samples
A
Rate-Zonal Centrifugation
16
Q
– A high density gradient is
used and cells are separated solely on differences in density (gradient will submerged)
o Uses high density gradient- cause the gradient to submerge at the bottom part of the tube, DNA will float on the top of the gradient
A
Isopycnic Centrifugation
17
Q
*WBCs separated from RBCs
*Virus separated from blood and body fluids-__ methods
A
centrifugation
18
Q
*Bacteria separated from contaminating materials-_ and _
A
(1% sodium dodecyl sulfate) and strong base (0.2 M NaOH)
19
Q
- Protease inhibitors-
A
phenylmethylsulfonyl fluoride (PMSF),
aminoethyl benzylsulfonyl fluoride,
tosyl lysine chloromethyl ketone,
tosylphenyl chloromethyl ketone,
EDTA,
benzamidine, and
peptide protease inhibitors.
20
Q
*Breaking the cell and nuclear membrane
- must take place in conditions that will not damage
the nucleic acid.
A
(cell lysis)
21
Q
depends on the protein target of extraction, sample
size, and experience in preparation methods.
*Examples:
A
LYSIS BUFFER
Triton X-100,
NP-40, Tween 20,
Octyl Glucoside,
Octyl Thioglucoside,
BIG CHAP, CHAPS, CHAPSO, Deoxycholate
22
Q
TARGET NUCLEIC ACID IS
PURIFIED
*Get rid of contaminating , and _
*Get read rid of other nucleic acid (DNA free of RNA or RNA
free of DNA).
*Concentration and purity of the sample can be determined.
A
proteins, carbohydrates, lipids, and cell debris
23
Q
___________________ - detergent, commonly used in separating bacteria from contaminating materials
A
1% sodium dodecyl sulfate (SDS)
24
Q
used as detergent material in molecular lab particularly in bacteria (for lysis of bacteria samples)
A
Triton X-100
25
TECHNIQUES TO REMOVE CONTAMINANTS AND DETERGENT FROM PROTEOMIC SAMPLES
- SALTING OUT
- ULTRAFILTRATION
- POLYETHYLENEIMINE
- ISOELECTRIC POINT
- THERMAL
- NONIONIC POLYMER
26
Saturation of salt to precipitate proteins. This may be in the form of an ammonium sulfate precipitation or sodium sulfate
SALTING OUT
27
Centrifugation at high-speed using molecular weight cutoff filter to remove contaminants
ULTRAFILTRATION
28
The use of a cationic polymer to precipitate nucleic acids in 1M NaCl, where proteins remain in supernatant (PEI must be removed before further analyses)
POLYETHYLENEIMINE
29
The pH is adjusted with mineral acid to the Pl of most proteins (pH 4-6) to precipitate neutral proteins
ISOELECTRIC POINT
30
Cell extracts are denatured with high temperatures causing them to precipitate. Stability is enhanced
THERMAL
31
- Polyethylene Glycol
- Concentration of PEG unique to the protein mixture is added. Due to the excluded volume principle and centrifugation, precipitated proteins are pelleted (PEG must be removed before further analyses)
NONIONIC POLYMER
32
Goal is to release nucleic acid from the cell for use in other procedures or other downstream experiments.
NUCLEIC ACID EXTRACTION METHODS
33
This is used to extract and purify RNA and DNA from biological samples that may contain tissue, blood, or other organic matter and prepare them for use.
NUCLEIC ACID EXTRACTION METHODS
34
starting point in a vast array of downstream application or experiments, the high quality of nucleic acids is a key factor for the success of the subsequent steps of the analysis.
NUCLEIC ACID EXTRACTION METHODS
35
Defined as a series of steps to obtain nucleic acid samples or materials of particular purity that are free of impurities and are suitable for different
downstream applications steps.
NUCLEIC ACID EXTRACTION
36
is to disintegrate the cell
envelope and to achieve maximum elimination of lipids and proteins to obtain
pure DNA and RNA.
NUCLEIC ACID EXTRACTION
37
NUCLEATED CELLS IN SUSPENSION
* * Two ways to isolate WBC:
1
2
* o Differential Density Gradient Centrifugation
* o Differential Lysis
38
*Whole blood or bone marrow mixed with isotonic saline, Overlayed with Ficoll.
* It is used in biology laboratories to separate blood into its components
DIFFERENTIAL DENSITY GRADIENT CENTRIFUGATION
39
is a highly branched sucrose polymer that does not
penetrate biological membran
Ficoll
40
is also known as__
*Takes advantage of the differences in the RBCs and WBCs.
* Incubation of whole blood or bone marrow in hypotonic
buffer or water will result in the lysis of RBC before the WBC
*The WBCs are then pelleted by centrifugation, leaving the
empty RBC membranes (ghosts) and hemoglobin, respectively, in suspension and solution
ISOLATION OF WBCS BY DIFFERENTIAL LYSIS
- differential extraction
41
–process in which DNA from two different type of cells can be extracted without mixing their content
* Differential extraction
42
__ must be dissociated
*__ samples are disrupted by grinding the frozen tissue in liquid nitrogen, homogenizing the tissue, or simply mincing the tissue using a scalpel.
Fresh or frozen tissue samples
Whole tissue
43
has to be deparaffinized by soaking in xylene (a mixture of three isomers of dimethylbenzene).
* Fixed embedded tissue
44
* Less toxic xylene substitutes, such as _, _, _, are also often used for this purpose.
Histosolve, AnatechPro-Par, or
ParaClear
45
*After xylene treatment, the tissue is usually rehydrated by
soaking it in decreasing concentrations of _.
ethanol
46
*The pellets are washed with ethanol After dewaxing, the
samples are subject to lysis and thorough homogenization
followed by incubation at _ for and _ for _
Subsequently, __is added for 48h digestion at 56°C.
65°C for 1h and 95°C for 15 min.
proteinase K
47
_______________ - salt concentration test
o To see if the RBC will rupture in different salt
concentration solutions
o Hypertonic saline solution
o Causes burst of RBC – will leave only WBC
Osmotic fragility
48
_______________ – concentration of solute outside is lower than the concentration inside; therefore, hypotonic buffer will move inside the cell causing it to swell and lyse
HYPOTONIC
49
______________ - Decreasing concentrations (90% to 80% to 70%)
Rehydration
50
________ - incr. concentration (70% to 80% to 90%)
Dehydration
51
* _______ and ______ having tough cell walls must be broken to allow the release of nucleic acid
* * By several enzyme products that digest cell wall polymers
* e.g., _ or _ are commercially available.
* * Alternatively, cell walls can be broken mechanically
* by grinding or by vigorously mixing with glass beads.
- Bacteria and fungi
- lyzozymeor zymolyase
52
Treatment with detergent _ and _ in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), and glucose can also break bacterial cell walls.
(1% sodium dodecyl sulfate) and strong base (0.2 M NaOH)
53
* Boiling in _,_ _ and_ after lysozyme treatment
releases DNA that can be immediately precipitated with alcohol.
8% sucrose, 8% Triton X-100 detergent, Tris buffer, and EDTA
54
Organisms that are hard to kill and have a tough cell wall (chitin in polysaccharide) - _______
FUNGI
55
____________________________ is used to breakdown tough cell walls of fungi
CETYL TRIMETHYL AMMONIUM BROMIDE (CTAB)
56
are less likely to damage chromosomal DNA and thus preferred for methods involving larger chromosomal targets as opposed to plasmid DNA.
Gentler enzymatic methods
57
DNA extracted with NaOH or boiling procedures is denatured (single-stranded) and may not be suitable for methods such as _ that require double-stranded DNA.
restriction enzyme analysis
58
Samples for molecular lab
o Whole blood
o Bone marrow
o Tissue
o Microorganism
59
a chelating resin that has a high affinity for polyvalent metal ions, can also be used to isolate fungi DNA quickly and efficiently.
* It is frequently used to release DNA from cells by a boiling treatment, at the same time protecting the DNA from the boiling effects with resin beads.
- CHELEX-100
60
Further purification of DNA from contaminating proteins, lipids, carbohydrates, and cell debris using a combination of high salt, low pH, and an organic mixture of phenol and chloroform.
DNA ORGANIC ISOLATION METHODS
61
When added to the hydrophilic cleared cell lysate, a biphasic emulsion forms.
PHENOL/CHLOROFORM
62
____________________- dissolves lipids and other hydrophobic components
hydrophobic layer on the bottom (organic phase)
63
Higher hydrophilic phase - hydrophilic layer on top dissolves DNA.
(aqueous phase)
64
___________________________ - collects a white precipitate of amphiphilic components, as well as cell debris
Middle interface between the two layers
65
* DNA is precipitated using _ or _ in a high concentration of salt (ammonium, potassium or
sodium acetate, or lithium or sodium chloride).
*Upon centrifugation, DNA forms a solid precipitate.
* The solid precipitate is collected by centrifugation.
* Excess salt is removed by rinsing the pellet in 70%
ethanol, centrifuging and discarding the ethanol
supernatant.
DNA PRECIPITATION
- ethanol (at 2:1 ratio) or isopropanol (at 1:1 ratio)
66
bottom part (white arrow). They are DNA and RNA/ nucleic acids
DNA pellet
67
process of adding buffer to DNA
Elution
68
solution of DNA + Tris buffer
Eluate
69
* DNA pellet is dissolved in rehydration buffer,
usually_ _ or _
10mM Tris, 1mM EDTA (TE), or water.
70
*Has been developed to avoid use of toxic reagents such as phenol.
* Initially, these methods did not provide the efficient recovery of clean DNA achieved with phenol extraction; however, newer methods
have proven to produce high quality DNA preparations in good yields.
*Proteins are selectively precipitated by low pH
and high salt conditions, while DNA is left in solution.
DNA INORGANIC ISOLATION METHODS
“SALTING OUT”
71
it makes us of low pH and high salt conditions.
“salting out”
72
are fast, easy-to-perform and economical.
- employ a simple bind-wash-elute process-
SILICA BASED METHOD
73
Nucleic acids bind to the silica membrane in the presence of __ -destroys /denatures
macromolecules-
Chaotropic Agent
74
_ and _ do not bind well to the column and residual traces are removed during alcohol
based wash steps, along with the salts-
After binding and washing, nucleic acids are selectively eluted under low-salt conditions, using _ or _
Polysaccharides and proteins
water or Tris
75
-based purification methods
also employ a bind wash-elute process
*-These workflows use magnetic beads or particles
functionalized with silica surfaces to allow selective
binding of DNA in the presence of high concentrations
of salt.
*-DNA bound to a magnetic bead can be easily
separated from the aqueous phase using a magnet;
thereby allowing rapid sample processing and fine
control of solution volumes.
MAGNETIC BEAD/ particle-based purification methods
76
_ in a slightly alkaline buffer or in ultra-pure water (optional).
Resolubilize DNA
77
Adding a _ agent to bind divalent cations (Mg2+and Ca2+) to stop
DNase activities.
chelating
78
Cellular and histone proteins bound to the DNA can be removed by adding a
_ or by having precipitated the proteins with sodium /ammonium acetate, or extracted them with a phenol chloroform mixture prior to the
DNA-precipitation
Protease
79
* is a broad-spectrum serine protease
*is commonly used in molecular biology to digest protein and remove contamination from preparations of nucleic acid.
*is also stable over a wide pH range of __
PROTEINASE K (ALSO PROTEASE K OR ENDOPEPTIDASE K)
- 4- 12
80
addition to nucleic acid
preparations rapidly degrades nucleases that might
otherwise degrade the DNA or RNA during
purification.
protease k
81
*First Approach
* (1) Sample preparation are homogenized by __
* (2) The homogenate is centrifuged at low speed to pellet intact_, _ and _
* (3) The mitochondria can be pelleted from the supernatant in a _
* (4) Mitochondria can be lysed with detergent and the lysate treated with _ to remove protein contaminants
* (5) Mitochondrial DNA can then be precipitated with _ and resuspended in water or the appropriate buffers for analysis
ISOLATION OF MITOCHONDRIAL DNA
- grinding on ice
- cells, nuclei, and cell debris
- second high-speed centrifugation
- proteinase
-cold ethanol
82
Second Approach
*The second approach is to isolate the _
*The preparation that contains mitochondrial DNA that can
be analyzed within the total DNA background by
_ or _
DNA
hybridization or PCR
83
*Requires STRICT precautions to avoid sample RNA degradation
*RNA especially labile
*Due to the ubiquitous presence of _
ISOLATION OF RNA
- RNAses
84
are naturally occurring enzymes that degrade RNA
- Common laboratory contaminant (From bacterial and human sources)
RNASES
85
Also released from cellular compartments during isolation of RNA from biological
samples
* can be difficult to inactivate
* o These enzymes are small proteins that can renature, even after autoclaving, and become active.
RNAses
86
* * Unlike DNAases, it must be eliminated or inactivated before isolation of
RNA
*o They remain active a wide range of temperatures
* e.gbelow -20C
RNASES
87
glassware must be baked for _ at _ to inactive the
RNAse
4-6 hours at 400C
88
80-90% of total RNA is __
* The most abundant of RNA in all cells
* Consists of two components, large and small, which are visualized by agarose gel electrophoresis
RNA in Agarose Gel
Electrophoresis
- ribosomal RNA (rRNA)
89
May be detected as a faint background underlying the rRNA detected by agarose gel
electrophoresis
* 2.5-5% is messenger RNA (mRNA)
90
_ and _ are also present in the total RNA
sample
Transfer RNA and Small nuclear RNAs
91
* Initial Step: Lyse/homogenize cells
*Add phenol: chloroform: __ to lysed sample and centrifuge
ORGANIC RNA EXTRACTION
isoamyl alcohol (25:24:1)
92
* Separation of molecules in solution or the mobile phase
* Base on differences in binding interaction with a ligand that is immobilized to
a stationary area, which is the solid phase
AFFINITY PURIFICATION OF RNA
93
MEASUREMENT OF NUCLEIC ACID QUALITY AND QUANTITY
- SPECTROPHOTOMETRY
- FLUORESCENCE
- ELECTROPHORESIS
94
• Method to measure how much chemical substance absorbs light by measuring the intensity of light as a beam of light passes through the sample
• Basic principle of spectrophotometry
• Each compound absorbs or transmit light over a certain range of wavelength.
SPECTROPHOTOMETRY
95
Emission of light by a substance that has absorbed light or other electromagnetic radiation.
FLUORESCENCE
96
General term that describes the migration and separation of charge particles or the ions under the influence of an electric field
ELECTROPHORESIS
97
* Often the RNA required for analysis is mRNA
* Accounting for only about 2.5-5% of the total RNA yield
* The majority of mRNA consist of mRNA from highly Expressed genes
* _can be used to purify mRNA from other RNAs
* Short stretches of single stranded DNA or RNA
ISOLATION OF POLY A (MESSENGER) RNA
- Oligo(dT) Oligonucleotide probes
