PCR Variants Flashcards
(151 cards)
1
Q
a process that involves a reverse transcriptase (RTase)
A
reverse transcription
2
Q
an enzyme that uses RNA as the template to make complementary DNA
A
reverse transcriptase (RTase)
3
Q
exact opposite of the naturally occuring DNA transcription in which RNA is synthesized using DNA as the template
A
reverse transcription
4
Q
in what temperature is needed to activate RTase
A
37°C
5
Q
primers used when the template is mRNA
A
Oligo (dT) primers
6
Q
oligo primers are attach when the template contains a ____
A
polyadenylated tail (poly-A tail)
7
Q
where does poly-A tail usually present?
A
mostly in eukaryotic RNA at the 3’
8
Q
how long is an oligo primer
A
18-base-long ss poly dT sequences
9
Q
primers that can be random hexamers or random decamers
A
random primers
10
Q
these primers are used if RNA is degraded RNA or has no poly-tail
A
random primers
11
Q
random primers are for what kind of RNA
A
prokaryotic or degraded RNA
12
Q
how long is a random primer
A
6 or 10-base long ss oligonucleotides of random sequences
13
Q
primers, RTase and standard PCR reagents are all combined in one tube which is then placed in a thermocycler
A
one-step RT-PCR
14
Q
RNA is converted to cDNA and then adding standard PCR reagents
A
two-step RT-PCR
15
Q
in two-step RT-PCR, when converting RNA and cDNA what reagents are added?
A
primer and RTase
16
Q
this is a technique used to amplify multiple target sequences in a single PCR reaction using multiple primer sets
A
multiplex PCR
17
Q
this method is used to detect deletions, polymorphisms, mutations, etc.
A
multiplex PCR
18
Q
this method is used to detect different viral, bacterial, and other pathogens in a single tube
A
multiplex PCR
19
Q
this method consumes less time and effort in obtaining results
A
multiplex PCR
20
Q
how many bands detect a positive result in gel electrophoresis of multiplex PCR?
A
3 bands
21
Q
how many bands detect a positive result in gel electrophoresis of conventional PCR?
A
1 solid band
22
Q
a modification of PCR that was
designed to improve sensitivity and specificity.
A
nested PCR
23
Q
this involves the use of two primer sets (external primers and nested primers) and two successive PCR reactions
A
nested PCR
24
Q
how many times does the PCR is repeated in nested PCR?
A
2 times
25
what is the sample used for the 2nd round in nested PCR?
the amplified product from the 1st round
26
this attributes to the high total
cycle number (20 cycles on 1st round plus 20 cycles on 2nd round)
increased sensitivity
27
this attributes to the added
primer (2 primers are used: outer primer and nested primer)
increased specificity
28
The target is amplified. Open the tube and use the first product for the 2nd round. Add the inner primer then amplify
traditional nested PCR
29
true or false: traditional nested PCR is prone to contamination and aerosol may enter the sample
true
30
contamination in traditional nested PCR can be avoided by physically separation the rounds of amplification mixture with?
a layer of wax or oil
31
in this type variant of nested PCR inner and out primers are combined in 1 reaction or tube
seminested asymmetrical PCR
32
In nested PCR, it can detect:
I. Rickettsia, Bartonella
II. Herpesvirus, enterovirus
III. Bacteremia
IV. M. tubercolosis in sputum
a. I, II, IV
b. I, II, III, IV
c, I, II, II
d. I only
B
33
It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time).
qPCR
34
In this variant of PCR, while detecting fluorescence, results are immediately displayed in the amplification plot.
Real Time or Real Time Quantitative PCR (qPCR)
35
These are used in qPCR to bind target DNA duringg annealing phase
fluorometric probes
36
true or false: The main advantage of qPCR over the traditional
PCR assays is that the starting DNA concentration can be determined with accuracy and high sensitivity.
true
37
this intercalates with any double-stranded DNA
non-specific fluorescent dyes
38
consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence.
sequence-specific DNA probes
39
this refers to the signal level during the initial cycles of PCR, usually cycles 3 to 15, in which there is little change in fluorescent signal
baseline
40
the baseline of the initial cycles of PCR is usually detected at?
3-15 cycles
41
it is the level of signal that reflects a statistically significant increase over the calculated baseline signal.
threshold
42
part of the graph in qPCR wherein the amplification starts
exponential phase
43
part of the graph in qPCR wherein the amplification is equal to the amount of reagents
linear phase
44
part of the graph in qPCR wherein all reagents have been consumed thus resulting a flat line
plateau phase
45
The cycle at which the amplification plot crosses the threshold
cycle threshold
46
How are the amplicons quantitated by the qPCR?
by using fluorescent signal
47
Patient A has a CT value of 10. Patient B has a CT value of 30. Both of them has SARS-Cov. Who among them has more virus in their body?
Patient A
CT value of Patient A is 10 which means an increase or there has been a presence of fluorescent signal at cycle 10. Higher concentration of virus so mas
maagang nag-cross sa threshold para ma detect na positive siya.
48
used for both specific and nonspecific detection of amplified product
ds DNA binding dye chemistry
49
used for specific PCR product detection
fluorophore-linked probes
50
a dye used in PCR that is toxic and carcinogenic
ethidium bromide
51
dye commonly used in PCR but unsuitable for high-resolution melt curve analysis (HRM)
SYBR Green
52
this dye is less inhibitory and is suitable for qPCR using fast cycling protocol
EvaGreen
53
This analysis consists of applying heat to the sample and monitoring the fluorescence emission during the process.
melting curve analysis
54
temperature needed in the melting curve analysis
from 50°C to 95°C
55
what does the melting curve analysis indicates if there are 2 peaks?
non-specific amplification
56
what does the melting curve analysis indicated if there only 1 peak appeared?
single specific product
57
what is the purpose of melting curve analysis?
a. to identify single specific product
b. to check if there are nonspecific amplification
c. both
d. none
B (pinaka main purpose is to check nonspecific product kasi it means may contamination or primer dimer)
58
these are small fluorescent molecules that are attached to oligonucleotides in order to function as probes
fluorophores
59
this method uses fluorescent and there are presence of two probes where one is the donor and other is the reporter
fluorescence resonance energy transfer (FRET)
60
in FRET, this causes the energy transfer
fluorescence
61
these are oligonucleotides that combine a primer and probe in a single molecule
primer probes
62
in what phase does the fluorescence emitted from primer-probes is detected and measured?
denaturation or extension
63
Include Scorpions, Amplifluor, and Light-Upon-eXtension (LUX)
hairpin primer-probes
64
in hairpin primer-probe, 5' identifies as what?
reporter
65
in hairpin primer-probe, 3' identifies as what?
quencher
66
in hairpin primer probe, this prevent reporter to emit fluorescence; known as a "blocker"
hexaethylene glycol
67
oligonucleotides with an attached-donor and/or acceptor fluorophore
probes
68
mechanism of action relies on the 5′–3′ exonuclease activity of Taq polymerase, which degrades the bound probe during amplification
hydrolysis probes
69
fluorescence emitted by binding hybridization probes can be measured
hybridization probes
70
in what phase does the hybridization probes occurs?
annealing or the extension phase
71
enables the absolute and reproducible quantification of target nucleic acids
digital PCR (dPCR)
72
the initial sample mix is partitioned into a large number of microscopic wells prior to the amplification step
digital PCR (dPCR)
73
what is the dPCR result if there is a presence of target
1
74
what is the dPCR result if there is no target
0
75
this variant of PCR is mostly used in detecting cancer or mutation
dPCR
76
This allows measurement of absolute quantification of amplicons
dPCR
77
this technique uses only one temperature all throughout the process
isothermal amplification
78
a specific, simple, rapid and cost-effective nucleic acid amplification method
Loop-mediated isothermal amplification (LAMP)
79
relies on auto-cycling strand displacement DNA synthesis which is carries out at a constant temperature water bath/heat block
LAMP-PCR
80
what DNA polymerase is used in LAMP-PCR?
Bacillus stearothermophylus
81
what is the reaction of LAMP to amplify the product?
40-60 minutes
82
what is the temperature needed for LAMP water bath?
60-65°C
83
for how many hours do you need to water bath in LAMP-PCR?
1 hour
84
what makes a positive result in LAMP-PCR turbid?
magnesium pyrophosphate
85
how many primers does the LAMP-PCR uses?
4-6 primers
a. FIP (Forward Inner Primer)
b. FP (Forward Primer)
c. BIP (Backward Inner Primer)
d. B3 (Outer Primer)
86
PCR for for RNA genomes or mRNA (gene expression)
reverse transcription PCR (RT-PCR)
87
PCR for multiple sets of primers or
multiple samples
multiplex PCR
88
PCR x2, confirms PCR specificity
nested PCR
89
real-time quantification, relative quantification
qPCR
90
PCR for absolute quantification
digital PCR
91
PCR for isothermal
LAMP-PCR
92
An isothermal amplification reaction, specific for target RNA sequences.
NASBA
93
enzyme that makes RNA from a promoter engineered in the primer region
T7 DNA-dependent RNA polymerase
94
enzyme that produces DNA from the RNA templates
AMV-RTase
95
enzyme that removes the RNA from cDNA without the heat-denaturing step
RNase H
96
enzyme with one carrying a T7 promoter sequence to amplify an RNA target sequence
P1/P2
P1 is the one carrying T7
97
who introduce NASBA in 1991
J. Compton
98
this primer is designed in such a manner that when it forms a double-stranded DNA, it codes for T7 RNA polymerase promoter site that helps in generating complementary RNA copies using a DNA template
Primer P1
99
this primer contains a 5' terminal T7 RNA polymerase promoter sequence
Primer P1
100
this enzyme elongates the primer, creating cDNA copy of the RNA template and forming and RNA/DNA hybrid
AMV-RTase
101
this enzyme recognized the hybrid as substrate and hydrolases the RNA portion of the hybrid, leaving ssDNA
RNase H
102
true or false: RNase destroys both RNA and DNA in RNA-DNA hybrids
false, it only destroys RNA
103
this primer anneals to the DNA from the RNA-DNA hybrid
Primer P2
104
this enzyme elongates P2 and makes the promoter portion of the DNA double-stranded and transcriptionally active
AMV-RT
105
what is produced during elongation of P2
dsDNA with target sequence and T7 promoter
106
what enzyme produces multiple copies of RNA transcripts complementary to the original target RNA
T7 RNA polymerase
107
What can you use to detect end products of NASBA?
I. fluorescent probes
II. gel electrophoresis
III. fluorescent dyes
IV. colorimetric assay
a. II, III, IV
b. I, II, III, IV
c. I, II, IV
d. I, and III only
C
108
true or false: the starting material of NASBA is double-stranded RNA and end product is single-stranded RNA
false, both starting and end are ALWAYS single-stranded RNA
109
in what temperature does NASBA is subjected to?
41°C
110
what is the amplification time of NASBA?
60-180 minutes (1-3 hours)
111
Fluorescence is detected in NASBA using:
a. beacons probes
b. micro plate ready
c. neither a and b
d. both a and b
D
112
isothermal process that permits 10^10-fold amplification
strand displacement amplification
113
how many minutes/hours does the strand displacement amplification process occurs?
15 minutes
114
Used to amplify and detect nucleic acids, like DNA or RNA, which relies on the ability of a restriction endonuclease and an exonuclease-deficient strand displacing DNA polymerase to generate copies of a target DNA sequence
strand displacement amplification
115
bumper sequence dispaces the target sequence
2 outer (bumper primers)
116
binds at the restriction site, and facilitate amplification of the target sequence
2 inner (SDA primers)
117
following are reagents required in strand displacement amplification except:
a. nicking endonucleases
b. exonucleases
c. SD DNA polymerase
d. all are required
e. none of the following are required
D
118
the goal of this step is to generate a new version of the DNA target to be amplified and involves modification of the target sequence
target generation
119
this step is where exponential copies of the target sequence previouslt made from the first step are synthesized
exponential target amplification
120
these are promising targets pf disease diagnosis, specifically cancer
MicroRNAs
121
SDA can diagnose food-borne pathogens such as?
Salmonella Enteritidis
122
where does exponential amplification starts in strand displacement amplification
nicking site
123
SDA temperature during initial denaturation
95°C
124
SDA amplification temperature during the last step
95°C
125
SDA amplification constant temperature
37°C
126
SDA detection method for pregnancy test, Hep, and Syphilis
lateral flow detection
127
A nucleic acid amplification method that can be used in diagnostic methods for DNA, RNA, proteins,
and cells
rolling circle amplification
128
what is the required temperature for rolling circle amplification
23-60℃
129
the small circle DNA is amplified
by polymerase extension of a primer
linear RCA
130
this has two sets of primers wherein one carry on extension and the second set hybridize with the first set of primers
exponential RCA
131
what is the result when the second set of primers hybridize with the product of the first set of primers
hyper-branching
132
the mechanism by which naturally
occurring circular plasmids and viral genomes are replicated.
RCR
133
what is the DNA polymerase that was tested during 1992 using circular DNAs
klenow fragment
134
In Andre Fire's study, what is the DNA polymerase that could extend a primer on partially randomized DNA circles
Escherichia coli
135
in what size can E. coli extend
42 and 52 nt in size and 180 nt in length
136
what is used as vectors in cells to encode biogically active RNAs via RCT?
circular oglionucleotides
137
what is used to encode and extend human telomere in RCT?
small DNA circles
138
this is a continuous synthesis produces a long ssDNA
with hundreds to thousands of repeat units
concatemers
139
template strand in linear DNA which is an oligonucleotide whose two ends are complementary to two target sequences that are connected by a linker sequence
padlock probe
140
what bacteriophage DNA polymerase is used to amplify DNA and RNA using circular template?
phi 29 DNA polymerase from Bacillus subtilis
141
this technique can amplify target template under the isothermal condition with outstanding rapidity and high sensitivity and specificty
polymerase spiral reaction
142
in what temperature PSR requires
60–65°C
143
how many primer/s and enzyme does PSR requires?
1 pair of primers and 1 enzyme
144
what is the PSR amplification time?
30-60 minutes
145
a single tube, highly sensitive, and selective isothermal amplification technique alternative to the polymerase chain reaction (PCR).
recombinase polymerase amplification (RPA)
146
what temperature is required in RPA?
37 and 42 degrees celcius
147
how many copies of DNA can RPA amplify in less than 20 minutes?
1-10 copies
148
what is used in RPA to be embedded into the helix?
recombinase protein uvsX
149
One of the most straightforward approaches for isothermal nucleic acid amplification, mimicking
an in vivo process of DNA replication
helicase-dependent amplification (HDA)
150
HDA optimum temperature range and time
60-65 degrees celcius; 30-120 mins
151
In HDA, this dye is used where you would see the result through your naked eye
Tetramethylbenzidine (TMB)
