Methods and PCR's

Question 1

Increase sensitivity of test system by making more copies of target, or probe, or attaching more signal producing molecules onto target

Answer 1

Amplification Methods

Question 2

__________________: PCR, RT-PCR, qPCR, and transcription based
amplification

Answer 2

Target amplification methods: PCR, RT-PCR, qPCR, and transcription based
amplification

Question 2

__________________: PCR, RT-PCR, qPCR, and transcription based
amplification

Answer 2

Target amplification methods: PCR, RT-PCR, qPCR, and transcription based
amplification

Question 3

__________________________: Ligase chain reaction (LCR), strand displacement
amplification (SDA), and QB replicase

Answer 3

Probe amplification methods: Ligase chain reaction (LCR), strand displacement
amplification (SDA), and QB replicase

Question 4

____________________________: Branched chain DNA (bDNA) amplification, hybrid
capture assay (HCA), and cleavage based amplification

Answer 4

Signal amplification methods: Branched chain DNA (bDNA) amplification, hybrid
capture assay (HCA), and cleavage based amplification

Question 5

Prototype method used to exponentially increase the amount of target DNA found in
a sample, making detection more sensitive

Answer 5

Polymerase Chain Reaction

Question 6

a. Sample DNA is denatured by heating to _________, _____ seconds.
b. Primers are added and the sample cooled to _______, _____ seconds to allow
primers to anneal (i.e., bind complementary sequences).

Answer 6

a. Sample DNA is denatured by heating to 94-96°C, 20-60 seconds.
b. Primers are added and the sample cooled to 50-70°C, 20-90 seconds to allow
primers to anneal (i.e., bind complementary sequences).

Question 7

c. _________________ (i.e., Tag polymerase) is added at ____ for _____ seconds to
extend primers and complete DNA synthesis of target sequence defined by primers. _____________ is a thermostable DNA polymerase isolated from the bacterium Thennus aquations.
d. A _____________ automatically changes temperatures and allows cycling to occur within a reaction vessel.

Answer 7

c. DNA polymerase (i.e., Tag polymerase) is added at 72°C for 10-60 seconds to
extend primers and complete DNA synthesis of target sequence defined by primers. Tag polymerase is a thermostable DNA polymerase isolated from the
bacterium Thennus aquations.
d. A thermocycler automatically changes temperatures and allows cycling to occur within a reaction vessel.

Question 8

e. Repeat the cycle ______ times to produce detectable levels of amplicons.
f. After* cycles, a 100% yield = 2*~2 (e.g., with 30 cycles, 228 = 2.68 X 108
amplicons from one template). The yield is not exactly 2X because the products
from the first two cycles are ____________ than the desired product

Answer 8

e. Repeat the cycle 20-30 times to produce detectable levels of amplicons.
f. After* cycles, a 100% yield = 2*~2 (e.g., with 30 cycles, 228 = 2.68 X 108
amplicons from one template). The yield is not exactly 2X because the products
from the first two cycles are slightly larger than the desired product

Question 9

___________________ can be detected by gel electrophoresis or labels can be introduced into the product using labeled primers or dNTP. Alternatively,
________________ or antibodies that recognize products that give off colorimetric,
fluorescent or ______________________ can be used.

Answer 9

Unlabeled products can be detected by gel electrophoresis or labels can be introduced into the product using labeled primers or dNTP. Alternatively,
labeled probes or antibodies that recognize products that give off colorimetric,
fluorescent or chemiluminescent signals can be used.

Question 10

: Known sample containing target sequence
1. Ensures that DNA polymerase enzyme is active
2. Buffer is optimal
3. Primers are annealing to correct sequence
4. Thermocycler is working properly

Answer 10

Positive control

Question 11

Reaction without DNA added to ensure that reagent mix is not contaminated with template or previously amplified PCR
products

Answer 11

Blank control or reagent control

Question 12

DNA sample known to lack target to ensure that
primers do not anneal to unintended sequence

Answer 12

Negative template control

Question 13

is a second primer set for a sequence unrelated to target sequence of interest but present in all samples tested. It can be performed in same tube or can be run as a duplicate sample.

Answer 13

Internal control or amplification control

Question 14

i. Ensures that DNA sample does not contain inhibitors and reaction mix is working properly
ii. Distinguishes between true negative (sample without target sequence) and false negative (amplification failure) results

Answer 14

Internal control or amplification control

Question 15

Estimates the amount of starting template (e.g., copy number/mL)

Answer 15

Quantitative Real-Time PCR (qPCR)

Question 16

______________________ are incorporated into PCR, thus generating fluorescently labeled amplicons. _______________ is generated as target copy number increases during amplification process.

Answer 16

Fluorescently labeled primers are incorporated into PCR, thus generating fluorescently labeled amplicons. Fluorescent signal is generated as target copy number increases during amplification process.

Question 17

____________________________
Analysis is done during the exponential phase of the reaction. The amount of
fluorescence generated is _____________________ to amount of starting template;
however, the time to its accumulation (the point when it crosses a predetermined
amount or threshold) is ________________, such that large amounts of target cross threshold early.

Answer 17

Quantitative Real-Time PCR (qPCR)
Analysis is done during the exponential phase of the reaction. The amount of
fluorescence generated is directly proportional to amount of starting template;
however, the time to its accumulation (the point when it crosses a predetermined
amount or threshold) is inversely proportional, such that large amounts of target
cross threshold early.

Question 18

____________________________
The cycle number at which fluorescence crosses a threshold is designated __
(threshold cycle). __ is _________________ to the amount of starting target. Thus, detection of a large amount of target is indicated by a ___________.

Answer 18

Quantitative Real-Time PCR (qPCR)
The cycle number at which fluorescence crosses a threshold is designated CT
(threshold cycle). CT is inversely proportional to the amount of starting target.
Thus, detection of a large amount of target is indicated by a lower CT value.

Question 19

Applications: Viral load, tumor load, and treatment monitoring

Answer 19

Quantitative Real-Time PCR (qPCR)

Question 20

____________ is a double-stranded DNA-binding dye that monitors accumulation
of PCR products as they are made (i.e., in real time). _____________ and/or _______________ are artifacts that generate fluorescence.

Answer 20

SYBR green is a double-stranded DNA-binding dye that monitors accumulation
of PCR products as they are made (i.e., in real time). Mispriming and/or primer
dimers are artifacts that generate fluorescence.

Question 21

________________: Probe of ssDNA oligomer is homologous to a specific sequence in the targeted region of the PCR template. As PCR product is
amplified, the probe is displaced and hydrolyzed, releasing fluorescence.

Answer 21

TaqMan probe: Probe of ssDNA oligomer is homologous to a specific sequence in the targeted region of the PCR template. As PCR product is
amplified, the probe is displaced and hydrolyzed, releasing fluorescence.

Question 22

____________________ measure accumulation of product at the ______________ in the PCR cycle. ______ is detected only when probes are bound to template before displacement by polymerase.

Answer 22

Molecular beacons measure accumulation of product at the annealing step in the PCR cycle. Signal is detected only when probes are bound to template before displacement by polymerase.

Question 23

______________________ are tailed with hairpin molecular beacons structure. In the presence of template, primer/probe is extended moving reporter molecule away from quencher molecule, which generates fluorescence.

Answer 23

Scorpion primer/probes are tailed with hairpin molecular beacons structure. In the presence of template, primer/probe is extended moving reporter molecule away from quencher molecule, which generates fluorescence.

Question 24

_______________________________ utilizes two probes: one with a 3’ fluorophore and one with a 5’ catalyst for the fluorescence that binds to adjacent target sites on the amplicon.

Answer 24

Fluorescent resonance energy transfer (FRET) utilizes two probes: one with a 3’ fluorophore and one with a 5’ catalyst for the fluorescence that binds to adjacent target sites on the amplicon.

Question 25

________________________________
RNA template is converted to a DNA copy (cDNA) by RNA-dependent DNA polymerase;
also known as reverse transcriptase. ____ product then serves as template to make millions of copies of target RNA sequence.

Answer 25

Reverse Transcription PCR (RT-PCR)
RNA template is converted to a DNA copy (cDNA) by RNA-dependent DNA polymerase;
also known as reverse transcriptase. cDNA product then serves as template to make millions of copies of target RNA sequence.

Question 26

Transcription-mediated amplification (TMA; GenProbe),
nucleic acid sequence-based
amplification (NASBA), and
self-sustaining sequence replication are examples of ______________________________.

Answer 26

Transcription-mediated amplification (TMA; GenProbe),
nucleic acid sequence-based
amplification (NASBA), and
self-sustaining sequence replication are examples of target amplification methods.

Question 27

___ is the target and primary product. Reactions are __________.

Answer 27

Transcription-Based Amplification Systems

RNA is the target and primary product. Reactions are isothermal.

Question 28

Applications: Direct detection of RNA viruses and RNA from other infectious agents, as
well as transcribed gene sequences

Answer 28

Transcription-Based Amplification Systems

Question 29

Ligase chain reaction (LCR) and strand displacement amplification (SDA) are
commercially available in the U.S.; QB replicase is available in Europe.

Answer 29

Probe Amplification Methods

Question 30

Number of target sequences in sample is not changed. Synthetic primers/probes
complementary to target nucleic acid are amplified.

Answer 30

Probe Amplification Methods

Question 31

Entire target sequence must be known to design four oligonucleotide primers. LCR primers bind adjacent to each other, separated by only one base.

Answer 31

Ligase chain reaction

Question 32

Enzymes: DNA polymerase synthesizes cDNA by extending primers, and DNA ligase seals gap between adjacent primers. Thermocycler needed

Answer 32

Ligase chain reaction

Question 33

Applications: Detect point mutations in known genes (e.g., beta-globin gene for
detection of sickle cell disease)

Answer 33

Ligase chain reaction

Question 34

Major amplification products are the probes/primers. Denaturation, then isothermal two-stage process of target generation followed
by exponential amplification phase

Answer 34

Strand displacement amplification

Question 35

First stage involves ______________; ______ and _____bind close to each
other. ______ have recognition site for a restriction enzyme (RE).

Answer 35

Strand displacement amplification

First stage involves target generation; primer and probe bind close to each
other. Probes have recognition site for a restriction enzyme (RE).

Question 36

_____________________
i. As the outer primers are extended, they displace the ______, which are
extended.
ii. A second set of complementary primers then binds to displaced probes,
and ___________________ extends the complementary primers, producing a double-stranded version of the probes.
iii. The ______ are the target DNA for the next stage of the process.

Answer 36

Strand displacement amplification
i. As the outer primers are extended, they displace the probes, which are
extended.
ii. A second set of complementary primers then binds to displaced probes,
and DNA polymerase extends the complementary primers, producing a double-stranded version of the probes.
iii. The probes are the target DNA for the next stage of the process.

Question 37

____________________
Second stage: ________________/____________________________.

Answer 37

Strand displacement amplification

Second stage: Exponential probe/target amplification phase.

Question 38

Addition of fluorogenic probe yields a fluorescent signal directly proportional to the amount of amplified probe/target.

Answer 38

Strand displacement amplification

Question 39

Application: SDA is the basis for the BD ProbeTec®ET for Mycobacterium tuberculosis, Chlamydia trachomastis, and Neisseria gonorrhoeae.

Answer 39

Strand displacement amplification

Question 40

__________________ is an RNA-dependent RNA polymerase from bacteriophage QB. The
target can be either denatured DNA or RNA.

Answer 40

QB replicase is an RNA-dependent RNA polymerase from bacteriophage QB. The
target can be either denatured DNA or RNA.

Question 41

Probe-bound template is amplified by mixing with QB replicase, which can
generate a billion RNA molecules/probes in less than __ minutes.

Answer 41

Probe-bound template is amplified by mixing with QB replicase, which can
generate a billion RNA molecules/probes in less than 15 minutes.

Question 42

Used for identification of infectious agents

Answer 42

QB replicase

Question 43

bDNA amplification,
HCA, and
cleavage-based amplification

Answer 43

Signal Amplification Methods

Question 44

______________________________

In _______________________, the number of target sequences does not change; instead, large amounts of signal are bound to the target sequences present in the sample, making detection more sensitive. These systems carry less risk of target contamination.

Answer 44

Signal Amplification Methods

In signal amplification systems, the number of target sequences does not change;
instead, large amounts of signal are bound to the target sequences present in the
sample, making detection more sensitive. These systems carry less risk of target contamination.

Question 45

____ is frequently used for quantification of target sequences in clinical samples,
especially viral load determinations.

Answer 45

bDNA is frequently used for quantification of target sequences in clinical samples,
especially viral load determinations.

Question 46

• A series of short oligomer probes captures target nucleic acids.
• Additional extender (or amplifier) probes bind target nucleic acids, and then
  multiple reporter molecules load target nucleic acid with signal.

Answer 46

bDNA

Question 47

____
Procedure: (1) _______________ (either RNA or DNA) is released from cells; (2) ______ is denatured; (3) ___________________ binds to capture probes, fixed to solid support; and (4) _____________ have sequences that are complementary to sequences in the target molecules and to sequences in the amplifier molecules. Binding of complementary sequences occurs.

Answer 47

bDNA
Procedure: (1) Target nucleic acid (either RNA or DNA) is released from cells; (2) DNA is denatured; (3) target nucleic acid binds to capture probes, fixed to solid support; and (4) extender probes have sequences that are
complementary to sequences in the target molecules and to sequences in the amplifier molecules. Binding of complementary sequences occurs.

Question 48

____
Reporter molecules labeled with alkaline phosphatase bind amplifer probes. _________ is added as substrate for alkaline phosphatase, and a
chemiluminescent signal is emitted.

Answer 48

bDNA
Reporter molecules labeled with alkaline phosphatase bind amplifer probes. Dioxetane is added as substrate for alkaline phosphatase, and a
chemiluminescent signal is emitted.

Question 49

______________ is marketed by Digene Diagnostics for detection of human papillomavirus,
hepatitis B virus, and cytomegalovirus.

Answer 49

HCA is marketed by Digene Diagnostics for detection of human papillomavirus,
hepatitis B virus, and cytomegalovirus.

Question 50

______
- Target DNA is released from cells and binds to _____ probes to form DNA/RNA hybrid molecules.
- DNA/RNA hybrid forms unique structure, which can be bound by antibodies to surface of microtiter well.

Answer 50

HCA
- Target DNA is released from cells and binds to ssRNA probes to form DNA/RNA hybrid molecules.
- DNA/RNA hybrid forms unique structure, which can be bound by antibodies to surface of microtiter well.

Question 51

______
- ____________________ are detected by binding alkaline
phosphataseconjugated anti-DNA/RNA hybrid antibodies in a typical “sandwich” assay.
- ________ is added and signal is measured

Answer 51

HCA
- Captured hybrids are detected by binding alkaline
phosphataseconjugated anti-DNA/RNA hybrid antibodies in a typical “sandwich” assay.
- Substrate is added and signal is measured

Question 52

_________________________: Based on activity of cleavase enzyme used in the Invader® assay (Third Wave Technologies, Inc.)

Answer 52

Cleavage-based amplification: Based on activity of cleavase enzyme used in the Invader® assay (Third Wave Technologies, Inc.)

Question 53

a. Detects target nucleic acid by a series of probes that bind to the target and overlap
b. ___________ recognizes overlapping sequence of DNA and cuts it.
c. During __________________, if probe and test sequences are complementary, ___ enzymatic cleavage reactions occur, resulting in a fluorescent signal.

Answer 53

Cleavage-based amplification

Question 54

Applications: Used in genetics, hemostasis (e.g., factor V Leiden mutation detection), and infectious disease

Answer 54

Cleavage-based amplification

Question 55

The order of nucleotides in DNA is determined.

Answer 55

DNA Sequencing

Question 56

Applications in the clinical laboratory include genotyping of microorganisms, detecting mutations, identifying human haplotypes, and determining
polymorphisms.

Answer 56

DNA Sequencing

Question 57

Automated sequences have replaced manual methods

Answer 57

DNA Sequencing

Question 58

________________, or sequencing by synthesis, is a commonly used method in automated DNA sequencers. In this method, a single strand of DNA to be sequenced is used as a template to enzymatically synthesize its complementary strand. Bases are added one at a time, and the instrument uses _____________________ to determine which base was actually added at each step.

Answer 58

Pyrosequencing, or sequencing by synthesis, is a commonly used method in automated DNA sequencers. In this method, a single strand of DNA to be sequenced is used as a template to enzymatically synthesize its complementary strand. Bases are added one at a time, and the instrument uses chemiluminescence to determine which base was actually added at each step.

Question 59

are the raw materials (e.g., primers, probes, antibodies, and other test components) used in “in-house” diagnostic assays.

Answer 59

Analyte-Specific Reagents (ASRs)

Question 60

______________________
They are classified I, II, and III. Most molecular tests for infectious disease and tissue typing are ______, and their performance is established during test validation.

Answer 60

Analyte-Specific Reagents (ASRs)

They are classified I, II, and III. Most molecular tests for infectious disease and tissue typing are class I, and their performance is established during test validation.

Question 61

Can be used in FISH, PCR, HCA, and microarray analysis

Answer 61

Analyte-Specific Reagents (ASRs)

Question 62

1. Methods: RFLP, ________________, and PCR
2. Targets: _____, SNP, ____, and mtDNA

Answer 62

Human Identity (DNA Polymorphisms)

1. Methods: RFLP, Southern blot, and PCR
2. Targets: STR, SNP, HLA, and mtDNA

Question 63

Applications: Forensics and paternity, post-stem cell/bone marrow engraftment testing,
linkage analysis of inherited (i.e., genetic) diseases, and tissue section identification

Answer 63

Human Identity (DNA Polymorphisms)

Question 64

Methods: HCA, ______, TMA, PCR, _____, RT-PCR, _____, SDA, and DNA sequencing
Targets: ___ and ____ rRNA, rDNA, housekeeping genes, toxin genes, antimicrobial
resistance genes, interspersed repetitive elements, strain-specific sequences, and
internal transcribed spacer elements

Answer 64

Detection, Identification, and Quantification of Microorganisms

Methods: HCA, NASBA, TMA, PCR, bDNA, RT-PCR, qPCR, SDA, and DNA sequencing
Targets: 16S and 23S rRNA, rDNA, housekeeping genes, toxin genes, antimicrobial
resistance genes, interspersed repetitive elements, strain-specific sequences, and
internal transcribed spacer elements

Question 65

Molecular epidemiology typing during outbreaks, genotyping, and drug resistance screening

Answer 65

Detection, Identification, and Quantification of Microorganisms

Question 66

a. ______________________ are highly reproducible and can discriminate between closely related organisms.
b. _____________________ analysis by _______________________ is commonly used for trace back studies during outbreaks of infectious
diseases.

Answer 66

a. Genotypic methods are highly reproducible and can discriminate between closely related organisms.
b. Chromosomal RFLP analysis by pulsed field gel electrophoresis is commonly used for trace back studies during outbreaks of infectious
diseases.

Question 67

Viral load (quantitative): bDNA, NASBA, Amplicor RT-PCR, and qPCR; especially for human immunodeficiency virus, ________________, hepatitis B virus, and ____________________________

Answer 67

Detection, Identification, and Quantification of Microorganisms

Question 68

Infectious disease testing of blood donor units: Units of blood are screened for a
number of bloodborne infectious agents using nucleic acid amplification tests.

Answer 68

Detection, Identification, and Quantification of Microorganisms

Question 69

Chromosomal abnormalities: Determine karyotype by ______ and _____________________

Answer 69

Chromosomal abnormalities: Determine karyotype by FISH and comparative genomic hybridization

Question 70

_________________________
Methods: DNA sequencing, PCR-RFLP, ____________, Southern blot, LCR, and __________________________

Answer 70

Single gene disorders
Methods: DNA sequencing, PCR-RFLP, linkage analysis, Southern blot, LCR, and capillary electrophoresis

Question 71

_____________
________________, ______ change; resulting in substitution of the arginine (R) at position 506 by glutamine (Q), R506Q

Answer 71

Single gene disorders
Factor V Leiden, G to A change; resulting in substitution of the arginine (R) at position 506 by glutamine (Q), R506Q

Question 72

1) ________: Loss of Mnll site
2) ________: Sequence-specific primers

Answer 72

1) PCR-RFLP: Loss of Mnll site
2) Single specific primer PCR (SSP-PCR): Sequence-specific primers

Question 73

Cystic fibrosis: _______, _________________________

Answer 73

Cystic fibrosis: CFTR gene, chloride channel membrane protein

Question 74

__________________________
a. Subset of STR with three bp repeating units that expand in length over generations.
b. ________________ is due to expanding copies of the CGG codon in the gene FMR-1 located on the X chromosome. It results in _________________ in males.
c. _______________ is due to CAG expansion at 4pl6.3.

Answer 74

Trinucleotide repeat expansion disorders
a. Subset of STR with three bp repeating units that expand in length over generations.
b. Fragile X syndrome is due to expanding copies of the CGG codon in the gene FMR-1 located on the X chromosome. It results in mental retardation in males.
c. Huntington disease is due to CAG expansion at 4pl6.3.

Question 75

Molecular oncology
Gene and chromosomal mutations in solid tumors can be detected by _________,
single-strand conformation polymorphism analysis, direct sequencing, _______________________, and _____. Translocation in __________________________

Answer 75

Molecular oncology

Gene and chromosomal mutations in solid tumors can be detected by SSP-PCR,
single-strand conformation polymorphism analysis, direct sequencing, immunohistochemical staining, and FISH. Translocation in hematologic malignancies