ANALYSIS AND CHARACTERIZATION OF NUCLEIC ACID AND PROTEIN Flashcards
1
Q
as related to genomics, is the process in which
two complementary single-stranded DNA and/or RNA molecules
bond together to form a double-stranded molecule
A
Hybridization
2
Q
target of southern blot
A
dna
3
Q
target of northern blot
A
rna
4
Q
target of western blot
A
protein
5
Q
target of southwestern blot
A
protein
6
Q
target of eastern blot
A
protein
7
Q
target of far eastern blot
A
protein
8
Q
method for molecular analysis of specific DNA sites within a
complex background without cloning that region
A
southern blot
9
Q
southern blot is named for ___
A
Edwin southern
10
Q
In the Southern blot, genomic DNA is isolated and cut with
__
A
restriction enzyme
11
Q
In the Southern blot, genomic DNA is isolated and cut with restriction enzymes.
The fragments are separated by gel
electrophoresis, denatured, and then transferred to a solid
support such as ___
A
nitrocellulose
12
Q
The first step in the Southern blot procedure is the __
A
digestion of test
13
Q
is carried out for an extended time to allow
complete cutting of all sites in the DNA
A
digestion
14
Q
If digestion is not carried out properly, it will produce an Incomplete cutting resulting in ___
A
Anomalous Pattern
15
Q
what does large aggregation of DNA at the top of electrophoresis means
A
indicates that the restriction enzyme was incomplete, preventing sample size analysis
16
Q
a smear located primarily in the lower region of the lane is a sign that the DNA is ___
A
degraded
17
Q
larger fragments are more efficiently denatured if they are __
before denaturation
A
depurinated
18
Q
large fragments, the gel is first soaked in ____, a process that removes purine bases from the sugar-phosphate back-bone. This will loosen up the larger fragments for more complete
denaturation.
A
dilute hydrogen chloride (HCl) solution
19
Q
- DNA is denatured by exposing the gel to a strong base such as
NaOH
A
DENATURATION
20
Q
a single stranded DNA avidly binds to the nitrocellulose membranes with a noncovalent but irreversible connection
A
membrane tyes
21
Q
nitrocellulose-based membranes can bind how many micrograms of nucleic acid
A
70-100
22
Q
Pure nitrocellulose has a high binding capacity for __
A
proteins as well as
nucleic acids
23
Q
this transfer method is simple and relatively inexpensive. No instruments
are required
A
Capillary transfer
24
Q
this transferring method uses electric current to move the DNA from
the gel to the membrane.
A
electrophoretic transfer
25
is a third method of DNA blotting
vacuum transfer
26
this involves incubating the membrane in the same buffer in which the probe will subsequently introduced or in a specially formulated prehybridization buffer solution
pre-hybridization
27
The buffer does not contain probe
t or f
true
28
Prehybridization buffer consists of such blocking
agents as
__
Denhardt solution and salmon sperm DNA
29
Denhardt solution components
ficoll
polyvinyl pyrrolidine
bovine serum albumin
30
The membrane is exposed to the prehybridization buffer at
the optimal hybridization temperature for ___ minutes to several hours, depending on the specifications in the protocol
30
31
a modification of the Southern blot technique,
was designed to investigate RNA structure and
quantity
northern blot
32
northern blot is used to
investigate levels of gene expression (transcription from DNA) and stability
investigate RNA structural abnormalities resulting from aberrations in synthesis or processing, such as alternative splicing
33
the splicing abnormalities is responsible for a number of diseases such as
beta thalassemia
familial isolated growth hormone deficiency
34
analysis of RNA structure and quantity indirectly reveals ___ in the regulatory or splicing signals in DNA
mutation
35
what are the uses of northern blot
nucleic acid isolation methods for RNA
rnase-free environment and must always be maintained
samples depending of the relative abundance of the transcript under study are applied directly to agarose gels
36
preferred agarose concentration in northern blot
0.8% to 1.5 %
37
preferred agarose concentration in southern blot
0-7%
38
polyacrylamide gels are used for
smaller transcripts of RNA
for analysis of viral gene expression
39
Gel electrophoresis of ____ is carried out under denaturing conditions for accurate transcript size assessment
RNA
40
is required for efficient transfer of the RNA from the gel to the membrane, as with the transfer of DNA in the southern blot
complete denaturation
41
how do we remove the denaturant after electrophoresis in northern blot
representative lanes are cut from the gel and soaked in `AMMONIUM ACETATE` to remove denaturant
42
in northern blot, after the electrophoresism the lanes that are soaked in ammonium acetate are now stained in ___ to assess quality and equivalent sample loading
acridine orange and ethidium bromide
43
DENATURANT example
formaldehyde
44
denaturant such as formaldehyde
- removal/acoomplised by rinsing the gel in
deionized water
45
a modification of southern blot technique wherein the the immobilized target is protein
western blot
46
serum, cell lysate or extract is separated by ___ in western blot
sodium dodecyl sulfate
polyacrylamide gel electrophoresis
isoelectric focusing
47
may also be used to
separate proteins into
subunits in western blot
dithiothreitol or 2-mercaptoethanol
48
Polyacrylamide concentrations in western blot
5% - 20%
49
In western blot, depending on the complexity of the protein and the quantity of the target protein how many protein is loaded per well
1-15 ug of protein
50
In western blot, before loading, the sample is treated with denaturant such as mixing ___
1:1 ratio of
0.04M tris HCL pH 6.8
0.1% SDS
51
denaturing gels
could affect _____ in such a way that they will not be able to bind with the labeled antibodies
epitopes (antigenic sites on
the protein)
52
how do we revive the denatured protein/denatured epitope
gel pre treatment with mild buffers such as
20% glycerol in 50mM tris-HCL pH 7.4
helps in renature proteins before transfer
53
it has high affinity for proteins and is easily treated with detergent with 5% dry milk to prevent binding of the primary antibody probe to the membrane itself (blocking) before hybridization
nitrocellulose
54
it prevents antibodies to attach in the nitrocellulose and helps to direct it to attach to the target protein
5% dry milk
55
Binding of proteins to nitrocellulose is
probably ___ because nonionic
detergents can remove proteins from the
membrane
hydrophobic
56
other membrane types used for protein blotting
PVDF (polyvinyl difluoride)
anion (DEAE) - Diethylaminoethyl
cation (CM) exchange cellulose -Carboxymethyl
57
don't forget that incubation with the primary antibody is for how many hrs
12-16 hrs
58
don't forget that after incubation with primary antibody for 12-16 hrs, the blot is washed in the same buffer and incubated with the ___
secondary antibody conjugated with enzyme
59
what will be the color that will be observed upon the secondary antibody incubation with conjugated enzyme
light color
60
after the secondary antibody conjugate or the 2nd incubation, the blot is __
washed again to remove excess secondary ab conjugate and the chemiluminescent or color signal is developed with the addition of substrate
61
example of radio isotope
p32 and p33
62
example of non radio isotope
biotin and digoxigenin
63
the probe for southern and northern blots is a ____ fragment of nucleic acid attached to a signal producing moiety
single-stranded
64
methods in probes for western blot
polyclonal antibody
monoclonal antibody
65
A labeled secondary antibody directed against the primary binding protein
used to visualize the protein band in electrophoresis
66
what are the nucleic acid probe
dna and rna
67
DNA probes are cloned on a bacterial plasmid and then isolated by ___
restriction enzyme digestion and gel purification
68
In vitro organic synthesis of nucleic acid of a predetermined
sequence (short, oligomeric probes)
dna probes
69
dna probes may also be synthesized using the __
PCR
70
The probe is constructed so that it has a complementary sequence to
the __
targeted gene
71
Double-stranded DNA probes must be denatured before use This is
usually accomplished by heating the probe via ___ or ____
heating the probe (e.g., 95°C, 10 to 15 min) in
hybridization solution
or
treating with 50% formamide/2 × SSC at a
lower temperature for a shorter time (e.g., 75°C, 5 to 6 m
72
in terms of stability, rna probe vs dna probe
dna probe - relatively stable
rna probe - not stable
73
the probe we use must be ___to the target
opposite
74
RNA probes are often made by
transcription of synthetic DNA
75
These probes are similar to DNA probes with equal or greater
binding affinity to complementary sequence
rna probes
76
RNA and DNA form a stronger helix than DNA/DNA
true or false
true
77
* RNA probes can be synthesized directly from
plasmid dna
transcription
78
what are the xample of predesigned systemns that are commercially available for the RNA probes (plasmid vector dna containing binding site for RNA polymerase, DDRP )
Salmonella bacteriophage SE6
~
Escherichia coli bacteriophage 3 NTE
79
other nucleic acid probe types, this on edo not have a negative charge hybridize more efficiently
peptide nucleic acid (PNA)
80
other nucleic acid probe types, this one lack the C2 and c3 ribose sugar bond found in ribonucleoside resulting in high flexibility
unlocked nucleic acids
81
this probe has an antibodies that bind specifically to the immobilized target protein
western blot probes
82
these are used for western blot probes
polyclonal and monoclonal
83
type of antibody used in western blot probes where the products has generalized response to a specific antigen ususally a peptide or peotein
polyclonal antibodies
84
polyclonal antibodies
Small molecules (__) attached to protein
carriers, carbohydrates, nucleic acids, and even to whole cells and tissue extracts can be used to
generate an antibody response.
haptens
85
Useful for immunoprecipitation methods and for western blots
polyclonal antibodies
86
western blot probe antibody
With their greater specificity, ___ can be used for
almost any procedure.
monoclonal antibodies
87
In western blot technology, ____antibodies can give a more robust
signal, especially if the target epitopes are partially lost during
electrophoresis and transfer.
polyclonal
88
__first demonstrated that spleen cells
from immunized mice could be fused with mouse myeloma
cells to form hybrid cells (hybridomas) that could grow in
culture and secrete antibodies
Kohler and Milstein
89
By cloning the __ (growing small cultures from single
cells), preparations of specific antibodies could be produced continuously
hybridomas
90
in cloning hybridomas, The clones could then be screened for antibodies that best react
with the target antigen
true or false
true
91
Probe labeling
traditional techniques in Southern analysis
involved the use of radioactive labeling using ___
32 P
92
This labeling was achieved by introduction of
nucleotides containing radioactive phosphorus to the probe
radioactive labeling using 32 P
93
In probe labeling, today, numerous medical laboratories have
adopted nonradioactive labeling methods
as a means to eliminate the risks and
expense of working with radiation
true or false
true
94
___ methods are based on indirect detection of a tagged nucleotide incorporated in or added to the probe.
Nonradioactive labeling
95
3 basic methods
end labelling
nick translation
random priming
96
1. end labeling
terminal transferase
t4 polymerase kinase
97
purpose of nick translation
for the probe to have a break
98
step wherein it generates new single stranded version of the probe with the incorporation of the labeled nucleotides
random priming
99
In random priming, the synthesis of these new strands is primed by oligomers of random sequences that are __
6 to 10 bases in length
100
RNA PROBES ARE TRANSCRIBED FROM
CLONED DNA OR
amplified DNA
101
the probes transcribed from DNA - RNA probes, These probes are labeled during their
synthesis with
radioactive,
biotinylated,
or digoxigenin-tagged nucleotides.
102
nucleic acid probe design
2 factors this affects
probe design
stringency
103
stringency consist of
temp
salt concent
concent of denaturation
104
With nucleic acids, the more optimal the hybridization conditions for a probe/target interaction, the more __the
probe
specific
105
longer probes has how many base pairs
500-5000 bp
106
shorter probes has how many base pairs
less than 500 bp
107
size of prove
It provides enhanced
precision while reducing
unwanted interference, as it
is less impacted by point
mutations or polymorphisms
longer probes
108
size of probe
difficult or expensive to synthesize
longer probes
109
size of probe
Easy to produce
cost effective
but is Affected by
mutation
shorter probe
110
less specific than longer
ones in Southern blotting
application
shorter probe
111
has a higher chance of being
repeated randomly in
unrelated regions of the
genome.
shorter probes
112
ideal size of probes
size
113
On the other hand, when it
comes to mutation analysis,
their ability to bind is easily
affected by even minor
alterations in a single base pair
within the desired binding
sequence
short probes
114
The protein probes used in western blot
applications may be labeled with ___ for
radioactive detection
35 S
115
For nonradioactive detection, western
protein probes are covalently bound to
an enzyme, usually __
horseradish
peroxidase (HRP) or alkaline phosphatase
116
in probe binding, when exposed to a light or color generating substrate, the enzyme will produce a detectable signal on the membrane
oks po
117
in probe binding, the unconjugated antibodies are detected with a after binding with ___ to the primary probe
conjugated secondary antibody
118
IN probe binding, the __ can affect its binding performance
probe sequence
119
* A probe containing complementary sequences within its structure will
undergo folding and binding to itself, resulting in __ with
the desired target for hybridization
competition
120
in probe binding, the probe folding or secondary structure is especially strong in sequences with HIgh ___ content
GC
121
probe folding will cause Decreasing the binding efficiency to the target sequence and,
therefore, the test sensitivity
true or false
true
122
is the combination of condition in which target is exposed to probe
Stringency
123
If the stringency level is set too high, the probe will not bind
to its target
t oir f
true
124
If the condition is set too low, it will bind to
unrelated target
125
SEVERAL FACTORS OF STRINGENCY
*Temperature of hybridization
*Salt concentration of hybridization
*Concentration of denaturant
126
advantage of p labeled probes
simple and sensitive detection
127
using the type of probe, after hybridization, the * unbound probe is washed off, and the blot is
exposed to light-sensitive film to detect the
fragments that are hybridized to the
radioactive probe
p labeled probes
128
non radioactive detection system
the probe is labeled with a nucleotide covalently attached to
either
DIGOXIGENIN or BIOTIN
129
Labelled Nucleotide is Incorporated into the Nucleotide Chain of the Probe by
* IN VITRO TRANSCRIPTION
* NICK TRANSLATION
* PRIMER EXTENSION
* ADDITION BY TERMINAL TRANSFERASE
130
conjugated to anti-digoxigenin antibody or
streptavidin
alkaline phosphatase
131
added to the reaction mix to bind to the
digoxigenin- or biotin-labeled probe: target
complex
alkaline phosphatase
132
HRP conjugates may also be used in this procedure
alkaline phosphatase
133
alkaline phosphatase, after conjugation, it will be bathed in a solution of substrate that when __produces a signal
dephosphorylated by AP or oxidized by HRP
134
bathing subtrate that will help in producing signal after the conjugation binding of alkaling phosphatase is
* DIOXETANE OR TETRAZOLIUM DYE DERIVATIVES
135
-substrates frequently used.
-generate chemiluminescent or color
signals
DIOXETANE OR TETRAZOLIUM DYE DERIVATIVES
136
The substrate used most often for chromogenic detection is a
mixture of
nitroblue tetrazolium (NBT) and 5-bromo-4-chloro3-indolyl phosphate (BCIP).
137
SUCCESSFUL BLOTTING METHOD IS A __ RATIO
HIGH SIGNAL-TO-NOISE
138
THE PROBE AND DETECTION SYSTEMS SHOULD
YIELD A __ SIGNAL
SPECIFIC AND ROBUST
139
__ are used with nonradioactive
detection systems to avoid nonspecific binding
of conjugate to the membrane
Blocking agents
140
how to interpret a result in hybridization
VISUALIZATION OF A “BAND”
141
__ CANNOT DETECT TINY DELETIONS OR
INSERTIONS OF NUCLEOTIDES OR SINGLE NUCLEOTIDE
DIFFERENCES UNLESS THEY AFFECT A RESTRICTION
ENZYME SITE
SOUTHERN BLOT
142
The
+__ is used to correct for any
differences in isolation or loading from sample to
sample
control transcript
143
