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MODX > ANALYSIS AND CHARACTERIZATION OF NUCLEIC ACID AND PROTEIN > Flashcards

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

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2
Q

target of southern blot

A

dna

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3
Q

target of northern blot

A

rna

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4
Q

target of western blot

A

protein

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5
Q

target of southwestern blot

A

protein

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6
Q

target of eastern blot

A

protein

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7
Q

target of far eastern blot

A

protein

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8
Q

method for molecular analysis of specific DNA sites within a
complex background without cloning that region

A

southern blot

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9
Q

southern blot is named for ___

A

Edwin southern

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10
Q

In the Southern blot, genomic DNA is isolated and cut with
__

A

restriction enzyme

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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

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12
Q

The first step in the Southern blot procedure is the __

A

digestion of test

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13
Q

is carried out for an extended time to allow
complete cutting of all sites in the DNA

A

digestion

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14
Q

If digestion is not carried out properly, it will produce an Incomplete cutting resulting in ___

A

Anomalous Pattern

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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

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16
Q

a smear located primarily in the lower region of the lane is a sign that the DNA is ___

A

degraded

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17
Q

larger fragments are more efficiently denatured if they are __
before denaturation

A

depurinated

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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

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19
Q
  • DNA is denatured by exposing the gel to a strong base such as
    NaOH
A

DENATURATION

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20
Q

a single stranded DNA avidly binds to the nitrocellulose membranes with a noncovalent but irreversible connection

A

membrane tyes

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21
Q

nitrocellulose-based membranes can bind how many micrograms of nucleic acid

A

70-100

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22
Q

Pure nitrocellulose has a high binding capacity for __

A

proteins as well as
nucleic acids

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23
Q

this transfer method is simple and relatively inexpensive. No instruments
are required

A

Capillary transfer

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24
Q

this transferring method uses electric current to move the DNA from
the gel to the membrane.

A

electrophoretic transfer

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25
Q

is a third method of DNA blotting

A

vacuum transfer

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26
Q

this involves incubating the membrane in the same buffer in which the probe will subsequently introduced or in a specially formulated prehybridization buffer solution

A

pre-hybridization

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27
Q

The buffer does not contain probe

t or f

A

true

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28
Q

Prehybridization buffer consists of such blocking
agents as
__

A

Denhardt solution and salmon sperm DNA

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29
Q

Denhardt solution components

A

ficoll
polyvinyl pyrrolidine
bovine serum albumin

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30
Q

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

A

30

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31
Q

a modification of the Southern blot technique,
was designed to investigate RNA structure and
quantity

A

northern blot

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32
Q

northern blot is used to

A

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

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33
Q

the splicing abnormalities is responsible for a number of diseases such as

A

beta thalassemia
familial isolated growth hormone deficiency

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34
Q

analysis of RNA structure and quantity indirectly reveals ___ in the regulatory or splicing signals in DNA

A

mutation

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35
Q

what are the uses of northern blot

A

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

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36
Q

preferred agarose concentration in northern blot

A

0.8% to 1.5 %

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37
Q

preferred agarose concentration in southern blot

A

0-7%

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38
Q

polyacrylamide gels are used for

A

smaller transcripts of RNA
for analysis of viral gene expression

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39
Q

Gel electrophoresis of ____ is carried out under denaturing conditions for accurate transcript size assessment

A

RNA

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40
Q

is required for efficient transfer of the RNA from the gel to the membrane, as with the transfer of DNA in the southern blot

A

complete denaturation

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41
Q

how do we remove the denaturant after electrophoresis in northern blot

A

representative lanes are cut from the gel and soaked in AMMONIUM ACETATE to remove denaturant

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42
Q

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

A

acridine orange and ethidium bromide

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43
Q

DENATURANT example

A

formaldehyde

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44
Q

denaturant such as formaldehyde
- removal/acoomplised by rinsing the gel in

A

deionized water

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45
Q

a modification of southern blot technique wherein the the immobilized target is protein

A

western blot

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46
Q

serum, cell lysate or extract is separated by ___ in western blot

A

sodium dodecyl sulfate
polyacrylamide gel electrophoresis
isoelectric focusing

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47
Q

may also be used to
separate proteins into
subunits in western blot

A

dithiothreitol or 2-mercaptoethanol

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48
Q

Polyacrylamide concentrations in western blot

A

5% - 20%

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49
Q

In western blot, depending on the complexity of the protein and the quantity of the target protein how many protein is loaded per well

A

1-15 ug of protein

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50
Q

In western blot, before loading, the sample is treated with denaturant such as mixing ___

A

1:1 ratio of

0.04M tris HCL pH 6.8
0.1% SDS

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51
Q

denaturing gels

could affect _____ in such a way that they will not be able to bind with the labeled antibodies

A

epitopes (antigenic sites on
the protein)

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52
Q

how do we revive the denatured protein/denatured epitope

A

gel pre treatment with mild buffers such as

20% glycerol in 50mM tris-HCL pH 7.4

helps in renature proteins before transfer

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53
Q

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

A

nitrocellulose

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54
Q

it prevents antibodies to attach in the nitrocellulose and helps to direct it to attach to the target protein

A

5% dry milk

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55
Q

Binding of proteins to nitrocellulose is
probably ___ because nonionic
detergents can remove proteins from the
membrane

A

hydrophobic

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56
Q

other membrane types used for protein blotting

A

PVDF (polyvinyl difluoride)
anion (DEAE) - Diethylaminoethyl
cation (CM) exchange cellulose -Carboxymethyl

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57
Q

don’t forget that incubation with the primary antibody is for how many hrs

A

12-16 hrs

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58
Q

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 ___

A

secondary antibody conjugated with enzyme

59
Q

what will be the color that will be observed upon the secondary antibody incubation with conjugated enzyme

A

light color

60
Q

after the secondary antibody conjugate or the 2nd incubation, the blot is __

A

washed again to remove excess secondary ab conjugate and the chemiluminescent or color signal is developed with the addition of substrate

61
Q

example of radio isotope

A

p32 and p33

62
Q

example of non radio isotope

A

biotin and digoxigenin

63
Q

the probe for southern and northern blots is a ____ fragment of nucleic acid attached to a signal producing moiety

A

single-stranded

64
Q

methods in probes for western blot

A

polyclonal antibody
monoclonal antibody

65
Q

A labeled secondary antibody directed against the primary binding protein

A

used to visualize the protein band in electrophoresis

66
Q

what are the nucleic acid probe

A

dna and rna

67
Q

DNA probes are cloned on a bacterial plasmid and then isolated by ___

A

restriction enzyme digestion and gel purification

68
Q

In vitro organic synthesis of nucleic acid of a predetermined
sequence (short, oligomeric probes)

A

dna probes

69
Q

dna probes may also be synthesized using the __

A

PCR

70
Q

The probe is constructed so that it has a complementary sequence to
the __

A

targeted gene

71
Q

Double-stranded DNA probes must be denatured before use  This is
usually accomplished by heating the probe via ___ or ____

A

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
Q

in terms of stability, rna probe vs dna probe

A

dna probe - relatively stable
rna probe - not stable

73
Q

the probe we use must be ___to the target

A

opposite

74
Q

RNA probes are often made by

A

transcription of synthetic DNA

75
Q

These probes are similar to DNA probes with equal or greater
binding affinity to complementary sequence

A

rna probes

76
Q

RNA and DNA form a stronger helix than DNA/DNA

true or false

A

true

77
Q
  • RNA probes can be synthesized directly from
A

plasmid dna
transcription

78
Q

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 )

A

Salmonella bacteriophage SE6
~
Escherichia coli bacteriophage 3 NTE

79
Q

other nucleic acid probe types, this on edo not have a negative charge hybridize more efficiently

A

peptide nucleic acid (PNA)

80
Q

other nucleic acid probe types, this one lack the C2 and c3 ribose sugar bond found in ribonucleoside resulting in high flexibility

A

unlocked nucleic acids

81
Q

this probe has an antibodies that bind specifically to the immobilized target protein

A

western blot probes

82
Q

these are used for western blot probes

A

polyclonal and monoclonal

83
Q

type of antibody used in western blot probes where the products has generalized response to a specific antigen ususally a peptide or peotein

A

polyclonal antibodies

84
Q

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.

A

haptens

85
Q

Useful for immunoprecipitation methods and for western blots

A

polyclonal antibodies

86
Q

western blot probe antibody

With their greater specificity, ___ can be used for
almost any procedure.

A

monoclonal antibodies

87
Q

In western blot technology, ____antibodies can give a more robust
signal, especially if the target epitopes are partially lost during
electrophoresis and transfer.

A

polyclonal

88
Q

__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

A

Kohler and Milstein

89
Q

By cloning the __ (growing small cultures from single
cells), preparations of specific antibodies could be produced continuously

A

hybridomas

90
Q

in cloning hybridomas, The clones could then be screened for antibodies that best react
with the target antigen

true or false

A

true

91
Q

Probe labeling

traditional techniques in Southern analysis
involved the use of radioactive labeling using ___

A

32 P

92
Q

This labeling was achieved by introduction of
nucleotides containing radioactive phosphorus to the probe

A

radioactive labeling using 32 P

93
Q

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

A

true

94
Q

___ methods are based on indirect detection of a tagged nucleotide incorporated in or added to the probe.

A

Nonradioactive labeling

95
Q

3 basic methods

A

end labelling
nick translation
random priming

96
Q
  1. end labeling
A

terminal transferase
t4 polymerase kinase

97
Q

purpose of nick translation

A

for the probe to have a break

98
Q

step wherein it generates new single stranded version of the probe with the incorporation of the labeled nucleotides

A

random priming

99
Q

In random priming, the synthesis of these new strands is primed by oligomers of random sequences that are __

A

6 to 10 bases in length

100
Q

RNA PROBES ARE TRANSCRIBED FROM

A

CLONED DNA OR
amplified DNA

101
Q

the probes transcribed from DNA - RNA probes, These probes are labeled during their
synthesis with

A

radioactive,
biotinylated,
or digoxigenin-tagged nucleotides.

102
Q

nucleic acid probe design

2 factors this affects

A

probe design
stringency

103
Q

stringency consist of

A

temp
salt concent
concent of denaturation

104
Q

With nucleic acids, the more optimal the hybridization conditions for a probe/target interaction, the more __the
probe

A

specific

105
Q

longer probes has how many base pairs

A

500-5000 bp

106
Q

shorter probes has how many base pairs

A

less than 500 bp

107
Q

size of prove

It provides enhanced
precision while reducing
unwanted interference, as it
is less impacted by point
mutations or polymorphisms

A

longer probes

108
Q

size of probe

difficult or expensive to synthesize

A

longer probes

109
Q

size of probe

Easy to produce
cost effective
but is Affected by
mutation

A

shorter probe

110
Q

less specific than longer
ones in Southern blotting
application

A

shorter probe

111
Q

has a higher chance of being
repeated randomly in
unrelated regions of the
genome.

A

shorter probes

112
Q

ideal size of probes

A

size

113
Q

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

A

short probes

114
Q

The protein probes used in western blot
applications may be labeled with ___ for
radioactive detection

A

35 S

115
Q

For nonradioactive detection, western
protein probes are covalently bound to
an enzyme, usually __

A

horseradish
peroxidase (HRP) or alkaline phosphatase

116
Q

in probe binding, when exposed to a light or color generating substrate, the enzyme will produce a detectable signal on the membrane

A

oks po

117
Q

in probe binding, the unconjugated antibodies are detected with a after binding with ___ to the primary probe

A

conjugated secondary antibody

118
Q

IN probe binding, the __ can affect its binding performance

A

probe sequence

119
Q
  • A probe containing complementary sequences within its structure will
    undergo folding and binding to itself, resulting in __ with
    the desired target for hybridization
A

competition

120
Q

in probe binding, the probe folding or secondary structure is especially strong in sequences with HIgh ___ content

A

GC

121
Q

probe folding will cause Decreasing the binding efficiency to the target sequence and,
therefore, the test sensitivity

true or false

A

true

122
Q

is the combination of condition in which target is exposed to probe

A

Stringency

123
Q

If the stringency level is set too high, the probe will not bind
to its target

t oir f

A

true

124
Q

If the condition is set too low, it will bind to

A

unrelated target

125
Q

SEVERAL FACTORS OF STRINGENCY

A

*Temperature of hybridization
*Salt concentration of hybridization
*Concentration of denaturant

126
Q

advantage of p labeled probes

A

simple and sensitive detection

127
Q

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

A

p labeled probes

128
Q

non radioactive detection system

the probe is labeled with a nucleotide covalently attached to
either

A

DIGOXIGENIN or BIOTIN

129
Q

Labelled Nucleotide is Incorporated into the Nucleotide Chain of the Probe by

A
  • IN VITRO TRANSCRIPTION
  • NICK TRANSLATION
  • PRIMER EXTENSION
  • ADDITION BY TERMINAL TRANSFERASE
130
Q

conjugated to anti-digoxigenin antibody or
streptavidin

A

alkaline phosphatase

131
Q

added to the reaction mix to bind to the
digoxigenin- or biotin-labeled probe: target
complex

A

alkaline phosphatase

132
Q

HRP conjugates may also be used in this procedure

A

alkaline phosphatase

133
Q

alkaline phosphatase, after conjugation, it will be bathed in a solution of substrate that when __produces a signal

A

dephosphorylated by AP or oxidized by HRP

134
Q

bathing subtrate that will help in producing signal after the conjugation binding of alkaling phosphatase is

A
  • DIOXETANE OR TETRAZOLIUM DYE DERIVATIVES
135
Q

-substrates frequently used.
-generate chemiluminescent or color
signals

A

DIOXETANE OR TETRAZOLIUM DYE DERIVATIVES

136
Q

The substrate used most often for chromogenic detection is a
mixture of

A

nitroblue tetrazolium (NBT) and 5-bromo-4-chloro3-indolyl phosphate (BCIP).

137
Q

SUCCESSFUL BLOTTING METHOD IS A __ RATIO

A

HIGH SIGNAL-TO-NOISE

138
Q

THE PROBE AND DETECTION SYSTEMS SHOULD
YIELD A __ SIGNAL

A

SPECIFIC AND ROBUST

139
Q

__ are used with nonradioactive
detection systems to avoid nonspecific binding
of conjugate to the membrane

A

Blocking agents

140
Q

how to interpret a result in hybridization

A

VISUALIZATION OF A “BAND”

141
Q

__ CANNOT DETECT TINY DELETIONS OR
INSERTIONS OF NUCLEOTIDES OR SINGLE NUCLEOTIDE
DIFFERENCES UNLESS THEY AFFECT A RESTRICTION
ENZYME SITE

A

SOUTHERN BLOT

142
Q

The
+__ is used to correct for any
differences in isolation or loading from sample to
sample

A

control transcript

143
Q
A

MODX (17 decks)

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