week7: NUCLEIC ACID ISOLATION ANDTECHNIQUESINEXTRACTION Flashcards
1
Q
a process of reducing a very pure nucleic acid, DNA and RNA, that is free from contaminants
A
extraction of nucleic acid
2
Q
goal of extraction of nucleic acid
A
to produce a very pure nucleic acid to use in nucleic acid testing such as PCR, blotting nucleic acid, and gel electrophoresis
3
Q
2 main methods used in extraction
A
precipitation and centrifugation1
4
Q
1869 the first one who isolated nucleic acid
A
friedrich miescher
5
Q
friedrich miescher
He discovered it when he was studying the chemical nature of the nuclei of the ___
A
white blood cell
6
Q
a method that friedrich miescher used to extract nucleic acid
A
precipitation
7
Q
the key discoverer of semi-conservative model of DNA
A
meselson and stahl
8
Q
what methods do meselson and stahl used to discover the semi conservicative model of DNA
A
centrifugation - density
9
Q
Nucleic acid extraction can be divided into three
steps:
A
breaking open tissues and cells
removing proteins/lipids/contaminants
transferring the nucleic acid to water or buffer solution that will preserve them without interfering with subsequent work
10
Q
breaking open tissues and cells is also called as
A
cell lysis
11
Q
mechanism to lyse a cell is
A
physical and chemical
12
Q
example of chemical lysing agent
A
on our experiment, we use of detergent
13
Q
what is the most commonly used
type of lysis buffer?
A
1-2% sodium dodecyl sulfate
14
Q
a reagent that will help to remove the protein contaminants
A
proteinase K
15
Q
a reagent that will help to remove the cell wall of gram negative bacteria
A
lysozymes
16
Q
a process called as removing contaminants is
A
homogenization or purification
17
Q
a reagent that will help to remove the peptidoglycan of gram positive bacteria
A
aminopeptidase
18
Q
the substep under the purification of nucleic acid
A
precipitation using alcohol
19
Q
why alcohol is used in precipitation of nucleic acid
A
insoluble in alcohol
20
Q
the purpose of the last step of nucleic acid extraction
A
for solubilization of the nucleic acid
21
Q
what is the media used in solubilization of the nucleic acid / resuspension
A
eluting buffer
- tris HCL
- tris acetate EDTA buffer
- molecular grade water
22
Q
the last step of extracting DNA is called is
A
elution
23
Q
the product of elution is called as
A
eluate
24
Q
CONSIDERATIONS FOR CHOOSING ISOLATION
METHOD
A
- specimen type - cellularity
- amount of sample and desired yield
- purity and size of isolate
- ease of operation and throughout
- cost and hazards
25
the DNA extracted from organism is called as
plasmid
26
3 main types of nucleic acid isolation
solid phase isolation
liquid phase isolation
chelex 100 method
27
the easiest method of isolation
solid phase
28
the most expensive method of isolation
chelex 100
29
the cheapest method of isolation
liquid phase isolation
30
liquid phase isolation uses phenol and chloroform. What are its downside
phenol can cause burns and chloroform is carcinogenic
31
this isolation method is cheap but has low yield of nucleic acid
liquid phase isolation
32
SAMPLES FOR MOLECULAR DIAGNOSTIC
the bacteria used for extraction is what gram type of bacteria
gram negative bacteria
33
SAMPLES FOR MOLECULAR DIAGNOSTIC
the fungi used for extraction is extracted in what part
yeast form and in hyphal structure
34
SAMPLES FOR MOLECULAR DIAGNOSTIC
the virus used for extraction is contaminated by ___
the dna of the host since they disintegrate themselves on the host cell
35
SAMPLES FOR MOLECULAR DIAGNOSTIC
the virus used for extraction is performed by this method ___
centrifugation
36
SAMPLES FOR MOLECULAR DIAGNOSTIC
the worms used for extraction is ___ prior to extraction
teased, grinded, minced
37
SAMPLES FOR MOLECULAR DIAGNOSTIC
the bacteria used for extraction is collected in what phase ___
harvested that grows in less than 24 hrs incubated
in mid logarithmic phase which means it's activately dividing and growing
38
SAMPLES FOR MOLECULAR DIAGNOSTIC
the blood used for extraction is ___
the one that is collected in tripotassium EDTA (EDTA K3) and acid-citrate-dextrose
purple and yellow top
39
SAMPLES FOR MOLECULAR DIAGNOSTIC
the forensic science we need for extraction is ___
chain of custody
40
why heparin is not used for extraction if using blood as sample
2 reason
- coextracted in the DNA extraction
- it inhibits enzymes such as reverse transcriptase and taq polymerase
41
SAMPLES FOR MOLECULAR DIAGNOSTIC
the tissue for extraction is ___
fresh and reserve
42
SAMPLES FOR MOLECULAR DIAGNOSTIC
the human cell used for extraction is ___
hair shaft, cells in nasopharyngeal and oropharyngeal
43
SAMPLES FOR MOLECULAR DIAGNOSTIC
the stool sample used for extraction is ___ prior extraction
digestion and decontamination
43
SAMPLES FOR MOLECULAR DIAGNOSTIC
the sediments used for extraction is ___ prior the extraction
centrifuge
44
SAMPLES FOR MOLECULAR DIAGNOSTIC
the plant used for extraction is extracted in what area
root and stem
45
MOSTCOMMONLYUSEDDNAEXTRACTION
PROCEDURES
liquid, solid, chelex 100
46
LIQUID PHASE ISOLATION METHOD
what are its 2 types
organic and inorganic
47
organic extraction of liquid-phase isolation method is through
phenol and chloroform which can cause burns and carcinogenic
48
inorganic extraction of liquid-phase isolation method is through the use of
proteinase K and salting out
49
salting out has what concentration of acid and salt
low pH and high salt concentratrion
50
Solid PHASE ISOLATION METHOD
what are its 2 types
manual method (silica-based method)
automated method (magnetic beads isolation)
51
the first type of solid phase extraction that is known as silica-based method uses specialized column with silica is called as
spin column
52
the two type of solid phase isolation (silica based and magnetic beads) follows the same process which are
adsorption and elution
53
the process which the solid phase method follows and is about binding and washing of nucleic acid
adsorption
54
the process which the solid phase method follows and is about transferring of nucleic acid for resuspension
elution
55
most preferred method and most commonly used isolation method
solid phase isolation
56
this type of isolation method is the most sensitive and is expensive
chelex 100
57
chelex 100 method uses ____ beads which is used enhance the attachment of polyvalent metal ions leaving the DNA in the solution
resin
58
this type of isolation of only small amount of sample or minimal sample
e.g. fungal extraction and forensic studies
chelex 100
59
the old method of nucleic acid isolation and is used to develop the semiconservative model of dna and is used for dna isolation
DENSITY GRADIENT CENTRIFUGATION STRATEGIES
60
DENSITY GRADIENT CENTRIFUGATION STRATEGIES
it divies the DNA into 2 bands called
main band
satellite band
61
DENSITY GRADIENT CENTRIFUGATION STRATEGIES
used ___ to allow the separation of the cells in the sample
preformed gradient
62
preformed gradient examples
sucrose and cesium chloride
63
the preformed gradient used in isolation of blood
sucrose = ficoll
64
preformed gradient initially used for DNA classification
cesium chloride
65
the main band in density gradient centrifugation strategies is rich in ___
adenine and thymine
66
the satellite band band in density gradient centrifugation strategies is rich in
cytosine and guanine
67
In separation of target cells/viruses
viruses are separated thrpugh
centrifugation
68
what is the reason why we need to prevent rbc contamination in wbc extraction
rbc will be hemolyzed in the process and the hemoglobin will inhibit the extraction of wbc
69
in older method, bacteria are separated from contaminating materials using
1% SDS and strong base solution (0.2M Sodium hydroxide NAOH )
70
this solution prevents the
Nucleic acid from enzymatic degradation of hydrolases
(e.g., phosphatases, glycosidases, and proteases)
7-9Murea, 2Mthiourea, or 2% SDS
71
disadvantage using 0.2 M NaOH (strong alkaline solution)
the nucleic acid will turn single stranded - denatured
72
3 main action of hydrolysis in nucleic acid
remove hydrogen bonds in between bases
destroy the phosphodiester bond
attacking the OH group
73
which one is more labile in terms of hydrolasis
rna or dna
rna as they have OH
74
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
75
Cell extracts are denatured with high temperatures causing them to precipitate. Stability is enhanced
Thermal
76
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
77
The choice of lysis buffer depends on the _____in
preparation methods.
protein target of extraction, sample size, and experience
78
a lysis buffer that is usually used in
boiling of bacteria
Triton X-100
79
Saturation of salt to precipitate protein
salting out
80
salting out may be in a form of __
ammonium sulfate or sodium sulfate
sodium acetate
81
the main mechanism of salt in salting out
removal of histone bounded in the DNA
82
Centrifugation at high speed using molecular weight
cutoff filter to remove contaminants
ultracentrifugation
83
isoelectric point uses what component
pH
84
a substance that is added in protein mixture that uses nonionic polymer technique
polyethylene glycol concentration
85
the preferred sample cell for WBC q
mononuclear cells
86
two ways to isolate WBC
differential density gradient centrifugation
differentitial lysis
87
In DIFFERENTIAL DENSITY GRADIENT CENTRIFUGATION, what is the component used
Ficoll, which is a sucrose
88
is a highly branched sucrose
polymer that does not penetrate biological
membranes.
Ficoll
89
It is used in biology laboratories to separate
blood into its components.
ficoll
90
Differential lysis is also known as
differential
extraction
91
process in which
DNA from two different type of cells can be
extracted without mixing their content.
differential extraction
92
this type of extraction method if nucleated cells that take advantage of the osmotic fragility of RBC and WBC
Differential lysis
93
Fixed embedded tissue has to be deparaffinized
by soaking in __
xylene
94
Wholetissue samples are disrupted by grinding
the frozen tissue in __, homogenizing
the tissue, or simply mincing the tissue using a scalpel.
liquid nitrogen
95
the ideal fixative in fixed tissue for dna extraction
10% neutral buffered formalin and acetone
96
the fixative to prevent in tissues
mercuric based fixative
Bouin's & B-5 Substitute Fixatives
carnoy's
97
Lesstoxic xylene substitutes, such as
___, are also often used for this
purpose
Histosolve, Anatech Pro-Par, or
ParaClear
98
the main type of extraction for tissue sample is the
proteinase K
99
are commercially
available for microorganism for extraction
Lysozyme or zymolyase
100
in the extraction of microorganism
Alternatively, cell walls can be broken mechanically.
○ ______
By grinding or by vigorously mixing with
glass beads.
101
we are using gentler enzymatic procedure involving lysozyme to extract nucleic acid from microorganism specially if the method we are using is ___
restriction fragment length polymorphism (abbreviated RFLP)
102
to remove the polysaccharide membrane in fungi, we are using
Cetyl trimethyl ammonium bromide (CTAB)
103
a chelating resin that has high affinity for polyvalent metal ions
chelex 100
104
purpose of magnesium and calcium in the extraction
are polyvalent metal ions that increases kinase enzyme
105
this isolation method that is for high amount of nucleic acid and is usually for fungal and forensic method
chelex 100
106
the use of phenol and chloroform method
DNA organic isolation method
107
3 layers of DNA organic isolation method formed
upper aqueous layer
interface layer
organic layer
108
the layer formed in dna organic isolation method where the DNA is found
upper aqueous layer
109
the layer formed in dna organic isolation method where the all cellular/white debri is found
interface layer
110
the layer formed in dna organic isolation method where the all the phenol and chloroform as well as lipid is found
organic layer
111
the most important layer in dna organic isolation method
upper aqueous layer
112
after getting the upper aqueous layer, what's next in the step?
precipitate the nucleic acid using alcohol
113
rules in aqueous and alcohol ratio
isopropanol - 1:1
ethanol 1:2 or twice the amount of ethanol
114
why do we need to increase the amount of ethanol if we're using such
it evaporates fast
115
purpose of the DNA precipitation
to form a pellet or a solid clump at the bottom f the microcentrifuge tube
116
how do you make sure that there's no alcohol left
wash in 75% alcohol
117
what is being precipitated in dna inorganic isolation method
protein, leaving the nucleic acid in the solution
118
this solid phase isolation that are fast, easy to perform and
economical.
silica-based method
119
Silica-based nucleic acid purification methods employ a simple __ process
bind-wash-elute
120
in the silica-based method, the __ will bind to the nucleic acid/dna
silica
121
in the silica-based method, what will happen to other components other than the dna
filtered out
122
usually used in extraction of RNA and is used in silica based method that disrupts macromolecules and DEGRADE all the RNASE present on the solutions
chaotropic agent
123
example of chaotropic agent
guanidine isothiocyanate (GITC)
124
principle of magnetic bead extraction
the positive charge magnet will selectively attract the negatively charge DNA in optimized conditions.
washing to remove the contaminants
wash the magnet to eluate in the solution
125
Is abroad-spectrum serine protease
proteinase K
126
__ is commonly used in molecular biology
to digest protein and remove contamination from preparations of nucleic acids.
Proteinase K
127
Proteinase K is also stable over a wide pH range of
4-12
128
isolation of mitochondrial dna
first and second approach
129
the approach in isolation of mitochondrial dna in which the sample must be only a hairshaft
second approach
130
in second approach , if we use a sample that is not a hairshaft will get what type of dna
total dna = nuclear dna + mtdna
131
to identify which is the mtdna among the total dna = nuclear dna + mtdna
pcr and hybridization procedure
132
to remove RNASE as well, aside from GITC, we can use
Diethyl pyrocarbonate (DEPC)
133
main purpose is to make water RNASE free
Diethyl pyrocarbonate (DEPC)
134
difference of ORGANIC RNA EXTRACTION to DNA organic extraction
addition of isoamyl alcohol
135
purpose of addition of isoamyl alcohol
remove foaming as it subsequently affect the extraction of RNA
136
how do we extract mRNA and not the total RNA
use the isolation of poly A (messenger) RNA
137
this process uses single stranded nucleic acid and has high binding to adenine
isolation of poly A (messenger) RNA
138
the short stretch of single stranded nucleic acid called as
oligo(dt) probe
139
this short probe can be used to purify mRNA from other RNA
oligo(DT ) probes
140
problem in isolation method of poly A (messenger) rna
final product is not that purified as no purification method is used
141
