Deck 11: Applications of Molecular Diagnostics

Question 1

The Gen-Probe AccuProbe system uses the following technology principle:

A) When two solutions are combined and hybridization occurs between the probe and the target, then the tube is centrifuged to detect the precipitated hybridized target and probes.
B) A single-stranded, chemiluminescent-labeled DNA probe is designed to hybridize to the target organism's ribosomal RNA (rRNA), forming a DNA:RNA duplex.
C) A double-stranded, bioluminescent-labeled DNA probe is designed to hybridize to the target organism's DNA, forming a DNA:DNA duplex.
D) A single-stranded, chemiluminescent-labeled RNA probe is designed to hybridize to the target organism's ribosomal RNA (rRNA), forming an RNA:RNA duplex.

Answer 1

B
A single-stranded,chemiluminescent labeled-DNA probe is designed to hybridize to the target organism's ribosomal RNA (rRNA),forming a DNA:RNA duplex.A detector called a liminometer is used to detect these labeled duplexes.The liminometer gives a reading in relative light units (RLU),and the RLU result for a suspicious culture is compared to a positive cutoff RLU value: any reading over the cutoff value is positive,and readings below are negative.

Question 2

The variables that affect the outcome of a given hybridization reaction include all of the following EXCEPT:

A) Organic acid concentration
B) Salt strength
C) pH
D) Temperature

Answer 2

A
These variables affecting the outcome of a given hybridization reaction include temperature,the nucleotide base composition of the probe,the length of the probe,the probe concentration,the degree of complementarity between the target and the probe,ionic strength (salt concentration),and pH.When one or more of these variables is not optimized for a hybridization assay,the hybrids may not efficiently form.

Question 3

Who received the Nobel Prize in Chemistry for inventing (polymerase chain reaction (PCR)?

A) Joseph Southern
B) Paul H. Gallo
C) Kary B. Mullins
D) Tim Klenow

Answer 3

C
The technique of synthetically synthesizing DNA was first described in 1971 by Khorana and colleagues,although Kary B.Mullins and others at the Cetus Corporation in California developed PCR into the current application in the early 1980s,eventually earning him the Nobel Prize in Chemistry for PCR.

Question 4

Many hybridization assay conditions are based on the expectation that a probe:

A) Will quickly react with the complementary target sequence.
B) Is not sterically hindered in the reaction
C) Has exact complementarity to the target nucleic acid
D) Will not require any oligonucleotides to attach to the target

Question 5

The target nucleic acid strand is:

A) The one to which the extra nucleic acid attaches
B) The DNA or RNA sequence unique to the organism of interest
C) The strand that is used as the oligonucleotide primer
D) The one that is labeled with a chromogen.

Question 6

What types of compounds are used for labeling probes?

A) Agar
B) Fluorescein
C) Digitoxin
D) Horseradish peroxidase

Question 7

The steps for performing the Northern blot test include all the following EXCEPT:

A) RNA is separated in an agarose gel.
B) The separated RNA is transferred from the agarose gel onto a membrane.
C) The membrane is then transferred to a gel so that the RNA can be immobilized in the gel.
D) The immobilized RNA is detected with a probe that hybridizes to the RNA species of interest.

Question 8

A Northern blot is used to detect:

A) Proteins
B) RNA
C) DNA
D) Lipids

Question 9

The function of the probe is to:

A) Form a duplex with every complementary sequence available in the reaction.
B) Make sure that the chromogenic or radioactive label is functional.
C) Break up the large oligonucleotides into smaller pieces so there will be more complementary pieces with which to pair.
D) Inhibit the formation of hybrids.

Question 10

Nucleic acid hybridization is:

A) Coupling of complementary single-stranded nucleic acid molecules
B) Mixing different pieces of DNA together and making a hybrid molecule
C) Attaching an RNA to an mRNA unit
D) Making RNA from a DNA template

Question 11

The principle of solid support hybridization (often called blotting)is:

A) The target sequence is part of the liquid support, and the probe, which is attached to solid support, hybridizes to the target.
B) All the target nucleic acids are gathered in one spot on the electrophoresis gel. Then all the probes are flooded in the same area and produce a blot of duplexes.
C) The target sequence is part of the solid support, and the probe, which is in solution, hybridizes to the target.
D) The probe is electrophoresed in agar, then transferred to filter paper. The antibody-based probe is then applied to the filter paper, and blots appear where there is binding.

Question 12

The steps for performing the Southern blot test include all the following EXCEPT:

A) After the labeled probe is hybridized to the specific target, the DNA is electrophoresed again, then dried and read.
B) The DNA is first digested with restriction enzymes.
C) Once digested, the DNA is separated and immobilized onto a solid membrane.
D) Labeled probe is hybridized to the specific DNA sequence.

Question 13

In a hybridization reaction,the pH affects:

A) The probe's nucleic acids so that they do not lose their hydrogens
B) The breakdown of the oligonucleotide pieces
C) The ability of the mismatches between the probe and the target to duplex
D) The stability of double-stranded nucleic acid molecules in solution

Question 14

The principle of in-solution hybridization is:

A) The hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells
B) The hybridization between an immobilized labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells
C) The hybridization between a labeled probe in solution and immobilized target nucleic acids
D) When two solutions are combined and hybridization occurs between the probe and the target, then the tube is centrifuged to detect the precipitated hybridized target and probes

Question 15

In situ hybridization,first described in 1969,is where:

A) Both RNA and DNA molecules can be detected simultaneously
B) Protein molecules can be detected in tissue specimens
C) A piece of tissue is soaked in a DNA solution, eliciting antinuclear antibodies in the tissue.
D) DNA or RNA can be detected directly in tissue with labeled probes.

Question 16

The probe is:

A) Immobilized on a solid support mechanism
B) The strand that is used as the oligonucleotide primer
C) The RNA portion that will act as the DNA replicase and allow replication to occur
D) Used to detect the target nucleic acid molecule

Question 17

How is the stability of a given hybrid calculated?

A) By calculating the number of certain types of bases in its structure
B) By calculating the length of the hybrid
C) By determining the melting temperature of a probe
D) By determining the ionic strength of the test solution

Question 18

Uses of probes include all of the following EXCEPT:

A) Microbial pathogen detection
B) Gene expression analysis
C) Abnormal WBC detection
D) Chromosomal translocations

Question 19

The Southern blot test separates:

A) RNA
B) Proteins
C) Lipids
D) DNA

Question 20

How do higher probe concentrations affect a given hybridization reaction?

A) Increase the temperature
B) Lower the reaction time
C) Lower the ionic strength
D) Increase the pH.

Question 21

Nested polymerase chain reaction (PCR)is very sensitive and specific because:

A) It uses more than one DNA polymerase to make three to four times the amount of PCR products than standard PCR.
B) The assay itself basically serves as a form of internal control and ensures specificity.
C) The assay will act as a form of external control, ensuring specificity and sensitivity.
D) The fluorochrome that is attached to the primers ensures specificity of the nucleotides that are attached to the template.

Question 22

Polymerase chain reaction (PCR)includes all the following steps EXCEPT:

A) Denaturation
B) Primer annealing
C) Primer extension
D) Amplification

Question 23

The most sensitive technique available for detecting and quantifying messenger RNA is:

A) Multiplex polymerase chain reaction (PCR)
B) Standard PCR
C) Nested PCR
D) Reverse-transcription PCR

Question 24

The advantages of real-time polymerase chain reaction (PCR)over standard PCR include all the following EXCEPT:

A) A positive result can be observed very quickly with RT-PCR.
B) RT-PCR does not use agarose gel.
C) RT-PCR does not accumulate hazardous waste.
D) RT-PCR uses open tubes like standard PCR.

Question 25

This piece of instrumentation is an integral part of the polymerase chain reaction (PCR)process.

A) Centrifuge
B) Thermal cycler
C) Electrophoretic chamber
D) Chemiluminescent detector

Question 26

PCR requires all of the following components EXCEPT:

A) DNA helicase
B) DNA polymerase
C) Oligonucleotides (primers)
D) Deoxynucleotide triphosphates

Question 27

The goal of the primer annealing step is to:

A) Hybridize oligonucleotide primers to the denatured, single-stranded target DNA strands.
B) Separate the target DNA so that the solution can be electrophoresed and a Northern blot performed.
C) Cut the native DNA into small pieces with a restriction enzyme.
D) Amplify exponentially over many cycles of these three reaction steps.

Question 28

The most common nucleic acid stain used after separation by agarose gel electrophoresis is:

A) Bromthymol blue
B) Bromcresol green
C) Ethidium bromide
D) Phenolphthalein

Question 29

The most commonly used polymerase is:

A) Escherichia coli 37 DNA polymerase
B) Taq DNA polymerase
C) Mg DNA polymerase
D) Dds DNA polymerase

Question 30

Multiplex PCR is good for:

A) Simultaneously detecting two or more different targets from one polymerase chain reaction (PCR) tube
B) Being the most sensitive method used to detect transfer RNA
C) Detecting PCR products at the lowest levels of any of the PCR tests
D) Using more than one DNA polymerase to make three to four times the amount of PCR products than standard PCR

Question 31

What is the principle of the 5' nuclease assay (Taqman)?

A) Fluorescent probe is mixed with labeled primers and a sandwich occurs where the fluorescent molecule is activated to fluorescence.
B) The Taq DNA polymerase extends from the primers and replicates the template to which the Taqman probe is annealed. The reporter dye is released by the 5' nuclease activity of the polymerase first, then the probe is released, thus increasing the fluorescence.
C) The Taq DNA polymerase extends from the primers and replicates the template to which the Taqman probe is annealed. Then the reporter dye is activated and the fluorescence increases with the number of hybrids that are produced.
D) A bioluminescent probe is attached to the 5' side of the primer. As the Taq DNA polymerase begins adding nucleotides to the growing chain, the last nucleotide added (because of steric hindrance) will be the bioluminescent probe. Once added, the probe will begin luminescing and the concentration of the hybrids is directly proportional to the amount of luminescence that is produced.

Question 32

NASBA stands for:

A) Nucleic acid sequence-based assay
B) Nucleic acid short-base amplification
C) Nucleic acid sequence-based amplification
D) Nucleotide amplification subsequent-base assay

Question 33

One drawback to polymerase chain reaction (PCR)is:

A) The process is easily contaminated.
B) The process takes a very long time to make the replications of DNA.
C) The results of the PCR test are very technique dependent, so that different technicians can get different results.
D) Patient care may be compromised if false-positive PCR results are generated.

Question 34

A Scorpion primer:

A) Extends the DNA polymerase's range
B) Is directed at a specific DNA target sequence
C) Uses a single oligonucleotide to prime a specific sequence and to detect accumulated polymerase chain reaction (PCR) product
D) Uses several oligonucleotides to prime sequences and acts as the beacon probe to detect hybrids

Question 35

What is the fluorescence resonance energy transfer (FRET)methodology?

A) The transfer of energy from a donor dye molecule to an acceptor dye molecule.
B) A fluorescent probe is mixed with labeled primers and a sandwich occurs where the fluorescent molecule is activated to fluorescent.
C) A light shines on the fluorescent probe label and it fluoresces.
D) A chromogenic substrate is mixed into solution, and when it combines with the Taq DNA polymerase, a fluorophore is formed and it fluoresces.

Question 36

In polymerase chain reaction (PCR),the target DNA is:

A) Complexed with RNA
B) Made using reverse transcriptase
C) Exponentially amplified over many cycles of PCR
D) Separated so that the solution can be electrophoresed and a Northern blot performed

Question 37

This cation is required for the proper function of the Taq DNA polymerase.

A) Ca
B) Na
C) Cl
D) Mg

Question 38

What is the purpose of the primer extension?

A) To cut the native DNA into small pieces with a restriction enzyme
B) To hybridize the oligonucleotide primers to the single stranded DNA pieces
C) To produce PCR products
D) To activate the DNA polymerase to form hybrids with the oligonucleotide primers

Question 39

During the denaturation step in polymerase chain reaction (PCR):

A) DNA polymerase extends the primers.
B) Oligonucleotide primers are hybridized to the single-stranded DNA.
C) Native DNA is cut into small pieces with a restriction enzyme.
D) The target double-stranded DNA is separated into single strands during the denaturation step.

Question 40

What chemical has been very successful in reducing carryover from polymerase chain reaction (PCR)assays?

A) Taq DNA polymerase
B) Uracil-N-glycosylate
C) Thymine
D) Bromthymol blue

Question 41

This method analyzes gene expression polymorphism by analyzing proteins.

A) Multilocus enzyme electrophoresis
B) Pulsed field gel electrophoresis
C) Restriction fragment length polymorphism (RFLP)
D) Multilocus sequence typing

Question 42

Accurate epidemiologic surveillance of specific organisms is needed for all the following reasons EXCEPT:

A) The rise of large numbers of antibiotic resistant isolates
B) Increases in the rates of toxin-producing bacteria
C) The spread of pathogenic microbes across the world
D) The increase in nosocomial infections

Question 43

All of the following nonamplified typing techniques are used to differentiate strains of an organism EXCEPT:

A) Multilocus sequence typing
B) Southern blotting
C) Plasmid profile analysis
D) Pulsed field gel electrophoresis

Question 44

All of the following amplified typing techniques are used to differentiate strains for an organism EXCEPT:

A) Random amplified polymorphic DNA
B) Pulsed field gel electrophoresis
C) Repetitive palindromic extragenic elements polymerase chain reaction (PCR)
D) Multilocus sequence typing

Question 45

This technique is a popular method of DNA fingerprinting.

A) Multilocus enzyme electrophoresis
B) Random amplified polymorphic DNA
C) Restriction fragment length polymorphism (RFLP)
D) Multilocus sequence typing

Question 46

This method is good for separating large DNA fragments in a low-percentage,low-melt agarose gel by an angled electrical field that periodically changes orientation.

A) Restriction fragment length polymorphism (RFLP)
B) Southern blot
C) Agarose gel electrophoresis
D) Pulsed field gel electrophoresis (PFGE)

Question 47

What is proteomics?

A) The study of proteins on a cellular level
B) The study of serum proteins
C) The study of proteins in genes
D) The study of the human genome

Question 48

The basis for this method is a microscopic grouping of DNA molecules attached to a solid support mechanism.Silicon chips,glass,or plastic have been used as the solid surfaces.What method is this?

A) Multilocus enzymes electrophoresis
B) Restriction fragment length polymorphism (RFLP)
C) Polymerase chain reaction (PCR)
D) DNA microarray

Question 49

When an organism's genome is sequenced,all of the following information can be obtained EXCEPT:

A) Cellular processes
B) Protein associations
C) Disease causing mechanisms
D) Interrelatedness of biologic activities

Question 50

What is the principle of restriction enzyme analysis of chromosomal DNA?

A) Plasmids are cut into small pieces with restriction enzymes. The resulting restriction fragment length polymorphism (RFLP) pattern is analyzed by agarose gel electrophoresis; there is transfer to a membrane, then use of a probe to identify specific sequences.
B) The chromosomal DNA is denatured, then annealed. The resulting hybrids are electrophoresed, transferred to a membrane, and then analyzed for a specific sequence.
C) DNA is extracted and isolated and digested with a restriction enzyme. The resulting RFLP pattern is analyzed by agarose gel electrophoresis and transferred to a membrane.
D) The serum specimen is electrophoresed, transferred to a membrane, digested, and then reacted with a probe to identify the target.