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MOLBIO MIDTERMS > PCR Variants > Flashcards

PCR Variants Flashcards

1
Q

a process that involves a reverse transcriptase (RTase)

A

reverse transcription

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2
Q

an enzyme that uses RNA as the template to make complementary DNA

A

reverse transcriptase (RTase)

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3
Q

exact opposite of the naturally occuring DNA transcription in which RNA is synthesized using DNA as the template

A

reverse transcription

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4
Q

in what temperature is needed to activate RTase

A

37°C

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5
Q

primers used when the template is mRNA

A

Oligo (dT) primers

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6
Q

oligo primers are attach when the template contains a ____

A

polyadenylated tail (poly-A tail)

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7
Q

where does poly-A tail usually present?

A

mostly in eukaryotic RNA at the 3’

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8
Q

how long is an oligo primer

A

18-base-long ss poly dT sequences

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9
Q

primers that can be random hexamers or random decamers

A

random primers

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10
Q

these primers are used if RNA is degraded RNA or has no poly-tail

A

random primers

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11
Q

random primers are for what kind of RNA

A

prokaryotic or degraded RNA

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12
Q

how long is a random primer

A

6 or 10-base long ss oligonucleotides of random sequences

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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

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14
Q

RNA is converted to cDNA and then adding standard PCR reagents

A

two-step RT-PCR

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15
Q

in two-step RT-PCR, when converting RNA and cDNA what reagents are added?

A

primer and RTase

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16
Q

this is a technique used to amplify multiple target sequences in a single PCR reaction using multiple primer sets

A

multiplex PCR

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17
Q

this method is used to detect deletions, polymorphisms, mutations, etc.

A

multiplex PCR

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18
Q

this method is used to detect different viral, bacterial, and other pathogens in a single tube

A

multiplex PCR

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19
Q

this method consumes less time and effort in obtaining results

A

multiplex PCR

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20
Q

how many bands detect a positive result in gel electrophoresis of multiplex PCR?

A

3 bands

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21
Q

how many bands detect a positive result in gel electrophoresis of conventional PCR?

A

1 solid band

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22
Q

a modification of PCR that was
designed to improve sensitivity and specificity.

A

nested PCR

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23
Q

this involves the use of two primer sets (external primers and nested primers) and two successive PCR reactions

A

nested PCR

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24
Q

how many times does the PCR is repeated in nested PCR?

A

2 times

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25
Q

what is the sample used for the 2nd round in nested PCR?

A

the amplified product from the 1st round

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26
Q

this attributes to the high total
cycle number (20 cycles on 1st round plus 20 cycles on 2nd round)

A

increased sensitivity

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27
Q

this attributes to the added
primer (2 primers are used: outer primer and nested primer)

A

increased specificity

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28
Q

The target is amplified. Open the tube and use the first product for the 2nd round. Add the inner primer then amplify

A

traditional nested PCR

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29
Q

true or false: traditional nested PCR is prone to contamination and aerosol may enter the sample

A

true

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30
Q

contamination in traditional nested PCR can be avoided by physically separation the rounds of amplification mixture with?

A

a layer of wax or oil

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31
Q

in this type variant of nested PCR inner and out primers are combined in 1 reaction or tube

A

seminested asymmetrical PCR

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32
Q

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

A

B

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33
Q

It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time).

A

qPCR

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34
Q

In this variant of PCR, while detecting fluorescence, results are immediately displayed in the amplification plot.

A

Real Time or Real Time Quantitative PCR (qPCR)

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35
Q

These are used in qPCR to bind target DNA duringg annealing phase

A

fluorometric probes

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36
Q

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.

A

true

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37
Q

this intercalates with any double-stranded DNA

A

non-specific fluorescent dyes

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38
Q

consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence.

A

sequence-specific DNA probes

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39
Q

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

A

baseline

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40
Q

the baseline of the initial cycles of PCR is usually detected at?

A

3-15 cycles

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41
Q

it is the level of signal that reflects a statistically significant increase over the calculated baseline signal.

A

threshold

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42
Q

part of the graph in qPCR wherein the amplification starts

A

exponential phase

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43
Q

part of the graph in qPCR wherein the amplification is equal to the amount of reagents

A

linear phase

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44
Q

part of the graph in qPCR wherein all reagents have been consumed thus resulting a flat line

A

plateau phase

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45
Q

The cycle at which the amplification plot crosses the threshold

A

cycle threshold

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46
Q

How are the amplicons quantitated by the qPCR?

A

by using fluorescent signal

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47
Q

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?

A

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.

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48
Q

used for both specific and nonspecific detection of amplified product

A

ds DNA binding dye chemistry

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49
Q

used for specific PCR product detection

A

fluorophore-linked probes

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50
Q

a dye used in PCR that is toxic and carcinogenic

A

ethidium bromide

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51
Q

dye commonly used in PCR but unsuitable for high-resolution melt curve analysis (HRM)

A

SYBR Green

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52
Q

this dye is less inhibitory and is suitable for qPCR using fast cycling protocol

A

EvaGreen

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53
Q

This analysis consists of applying heat to the sample and monitoring the fluorescence emission during the process.

A

melting curve analysis

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54
Q

temperature needed in the melting curve analysis

A

from 50°C to 95°C

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55
Q

what does the melting curve analysis indicates if there are 2 peaks?

A

non-specific amplification

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56
Q

what does the melting curve analysis indicated if there only 1 peak appeared?

A

single specific product

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57
Q

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

A

B (pinaka main purpose is to check nonspecific product kasi it means may contamination or primer dimer)

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58
Q

these are small fluorescent molecules that are attached to oligonucleotides in order to function as probes

A

fluorophores

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59
Q

this method uses fluorescent and there are presence of two probes where one is the donor and other is the reporter

A

fluorescence resonance energy transfer (FRET)

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60
Q

in FRET, this causes the energy transfer

A

fluorescence

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61
Q

these are oligonucleotides that combine a primer and probe in a single molecule

A

primer probes

62
Q

in what phase does the fluorescence emitted from primer-probes is detected and measured?

A

denaturation or extension

63
Q

Include Scorpions, Amplifluor, and Light-Upon-eXtension (LUX)

A

hairpin primer-probes

64
Q

in hairpin primer-probe, 5’ identifies as what?

A

reporter

65
Q

in hairpin primer-probe, 3’ identifies as what?

A

quencher

66
Q

in hairpin primer probe, this prevent reporter to emit fluorescence; known as a “blocker”

A

hexaethylene glycol

67
Q

oligonucleotides with an attached-donor and/or acceptor fluorophore

A

probes

68
Q

mechanism of action relies on the 5′–3′ exonuclease activity of Taq polymerase, which degrades the bound probe during amplification

A

hydrolysis probes

69
Q

fluorescence emitted by binding hybridization probes can be measured

A

hybridization probes

70
Q

in what phase does the hybridization probes occurs?

A

annealing or the extension phase

71
Q

enables the absolute and reproducible quantification of target nucleic acids

A

digital PCR (dPCR)

72
Q

the initial sample mix is partitioned into a large number of microscopic wells prior to the amplification step

A

digital PCR (dPCR)

73
Q

what is the dPCR result if there is a presence of target

A

1

74
Q

what is the dPCR result if there is no target

A

0

75
Q

this variant of PCR is mostly used in detecting cancer or mutation

A

dPCR

76
Q

This allows measurement of absolute quantification of amplicons

A

dPCR

77
Q

this technique uses only one temperature all throughout the process

A

isothermal amplification

78
Q

a specific, simple, rapid and cost-effective nucleic acid amplification method

A

Loop-mediated isothermal amplification (LAMP)

79
Q

relies on auto-cycling strand displacement DNA synthesis which is carries out at a constant temperature water bath/heat block

A

LAMP-PCR

80
Q

what DNA polymerase is used in LAMP-PCR?

A

Bacillus stearothermophylus

81
Q

what is the reaction of LAMP to amplify the product?

A

40-60 minutes

82
Q

what is the temperature needed for LAMP water bath?

A

60-65°C

83
Q

for how many hours do you need to water bath in LAMP-PCR?

A

1 hour

84
Q

what makes a positive result in LAMP-PCR turbid?

A

magnesium pyrophosphate

85
Q

how many primers does the LAMP-PCR uses?

A

4-6 primers

a. FIP (Forward Inner Primer)
b. FP (Forward Primer)
c. BIP (Backward Inner Primer)
d. B3 (Outer Primer)

86
Q

PCR for for RNA genomes or mRNA (gene expression)

A

reverse transcription PCR (RT-PCR)

87
Q

PCR for multiple sets of primers or
multiple samples

A

multiplex PCR

88
Q

PCR x2, confirms PCR specificity

A

nested PCR

89
Q

real-time quantification, relative quantification

A

qPCR

90
Q

PCR for absolute quantification

A

digital PCR

91
Q

PCR for isothermal

A

LAMP-PCR

92
Q

An isothermal amplification reaction, specific for target RNA sequences.

A

NASBA

93
Q

enzyme that makes RNA from a promoter engineered in the primer region

A

T7 DNA-dependent RNA polymerase

94
Q

enzyme that produces DNA from the RNA templates

A

AMV-RTase

95
Q

enzyme that removes the RNA from cDNA without the heat-denaturing step

A

RNase H

96
Q

enzyme with one carrying a T7 promoter sequence to amplify an RNA target sequence

A

P1/P2

P1 is the one carrying T7

97
Q

who introduce NASBA in 1991

A

J. Compton

98
Q

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

A

Primer P1

99
Q

this primer contains a 5’ terminal T7 RNA polymerase promoter sequence

A

Primer P1

100
Q

this enzyme elongates the primer, creating cDNA copy of the RNA template and forming and RNA/DNA hybrid

A

AMV-RTase

101
Q

this enzyme recognized the hybrid as substrate and hydrolases the RNA portion of the hybrid, leaving ssDNA

A

RNase H

102
Q

true or false: RNase destroys both RNA and DNA in RNA-DNA hybrids

A

false, it only destroys RNA

103
Q

this primer anneals to the DNA from the RNA-DNA hybrid

A

Primer P2

104
Q

this enzyme elongates P2 and makes the promoter portion of the DNA double-stranded and transcriptionally active

A

AMV-RT

105
Q

what is produced during elongation of P2

A

dsDNA with target sequence and T7 promoter

106
Q

what enzyme produces multiple copies of RNA transcripts complementary to the original target RNA

A

T7 RNA polymerase

107
Q

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

A

C

108
Q

true or false: the starting material of NASBA is double-stranded RNA and end product is single-stranded RNA

A

false, both starting and end are ALWAYS single-stranded RNA

109
Q

in what temperature does NASBA is subjected to?

A

41°C

110
Q

what is the amplification time of NASBA?

A

60-180 minutes (1-3 hours)

111
Q

Fluorescence is detected in NASBA using:

a. beacons probes
b. micro plate ready
c. neither a and b
d. both a and b

A

D

112
Q

isothermal process that permits 10^10-fold amplification

A

strand displacement amplification

113
Q

how many minutes/hours does the strand displacement amplification process occurs?

A

15 minutes

114
Q

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

A

strand displacement amplification

115
Q

bumper sequence dispaces the target sequence

A

2 outer (bumper primers)

116
Q

binds at the restriction site, and facilitate amplification of the target sequence

A

2 inner (SDA primers)

117
Q

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

A

D

118
Q

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

A

target generation

119
Q

this step is where exponential copies of the target sequence previouslt made from the first step are synthesized

A

exponential target amplification

120
Q

these are promising targets pf disease diagnosis, specifically cancer

A

MicroRNAs

121
Q

SDA can diagnose food-borne pathogens such as?

A

Salmonella Enteritidis

122
Q

where does exponential amplification starts in strand displacement amplification

A

nicking site

123
Q

SDA temperature during initial denaturation

A

95°C

124
Q

SDA amplification temperature during the last step

A

95°C

125
Q

SDA amplification constant temperature

A

37°C

126
Q

SDA detection method for pregnancy test, Hep, and Syphilis

A

lateral flow detection

127
Q

A nucleic acid amplification method that can be used in diagnostic methods for DNA, RNA, proteins,
and cells

A

rolling circle amplification

128
Q

what is the required temperature for rolling circle amplification

A

23-60℃

129
Q

the small circle DNA is amplified
by polymerase extension of a primer

A

linear RCA

130
Q

this has two sets of primers wherein one carry on extension and the second set hybridize with the first set of primers

A

exponential RCA

131
Q

what is the result when the second set of primers hybridize with the product of the first set of primers

A

hyper-branching

132
Q

the mechanism by which naturally
occurring circular plasmids and viral genomes are replicated.

A

RCR

133
Q

what is the DNA polymerase that was tested during 1992 using circular DNAs

A

klenow fragment

134
Q

In Andre Fire’s study, what is the DNA polymerase that could extend a primer on partially randomized DNA circles

A

Escherichia coli

135
Q

in what size can E. coli extend

A

42 and 52 nt in size and 180 nt in length

136
Q

what is used as vectors in cells to encode biogically active RNAs via RCT?

A

circular oglionucleotides

137
Q

what is used to encode and extend human telomere in RCT?

A

small DNA circles

138
Q

this is a continuous synthesis produces a long ssDNA
with hundreds to thousands of repeat units

A

concatemers

139
Q

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

A

padlock probe

140
Q

what bacteriophage DNA polymerase is used to amplify DNA and RNA using circular template?

A

phi 29 DNA polymerase from Bacillus subtilis

141
Q

this technique can amplify target template under the isothermal condition with outstanding rapidity and high sensitivity and specificty

A

polymerase spiral reaction

142
Q

in what temperature PSR requires

A

60–65°C

143
Q

how many primer/s and enzyme does PSR requires?

A

1 pair of primers and 1 enzyme

144
Q

what is the PSR amplification time?

A

30-60 minutes

145
Q

a single tube, highly sensitive, and selective isothermal amplification technique alternative to the polymerase chain reaction (PCR).

A

recombinase polymerase amplification (RPA)

146
Q

what temperature is required in RPA?

A

37 and 42 degrees celcius

147
Q

how many copies of DNA can RPA amplify in less than 20 minutes?

A

1-10 copies

148
Q

what is used in RPA to be embedded into the helix?

A

recombinase protein uvsX

149
Q

One of the most straightforward approaches for isothermal nucleic acid amplification, mimicking
an in vivo process of DNA replication

A

helicase-dependent amplification (HDA)

150
Q

HDA optimum temperature range and time

A

60-65 degrees celcius; 30-120 mins

151
Q

In HDA, this dye is used where you would see the result through your naked eye

A

Tetramethylbenzidine (TMB)

MOLBIO MIDTERMS (2 decks)

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