MULTIPLE CHOICE
1. 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 t
...
MULTIPLE CHOICE
1. 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.
A
Conceptually, nucleic acid hybridization refers to the formation of hydrogen bonds between nucleotides of single-stranded DNA or RNA molecules that are complementary. This forms a stable double-stranded nucleic acid molecule under the right conditions.
REF: 227 OBJ: Level 1: Recall
2. The target nucleic acid strand is the:
a. one to which the extra nucleic acid attaches.
b. DNA or RNA sequence unique to the organism of interest.
c. strand that is used as the oligonucleotide primer.
d. one that is labeled with a chromogen.
B
This is the portion of DNA that we are interested in identifying. The target nucleic acid molecule is typically either immobilized on a solid support mechanism or suspended in solution.
REF: 227 OBJ: Level 1: Recall
3. 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.
D
The probe is usually a single-stranded DNA or RNA oligonucleotide that is labeled with an attached report chemical or radionucleotide that can be detected either visually, by film, or by an instrument. The probe is produced synthetically to detect a specific target nucleic acid sequence of a given microorganism.
REF: 228 OBJ: Level 1: Recall
4. Common uses of probes include all of the following except:
a. microbial pathogen detection.
b. gene expression analysis.
c. abnormal WBC detection.
d. chromosomal translocations.
C
Probes have many uses including the detection of microbial pathogens in many different types of samples, gene expression analysis, gene rearrangements and chromosomal translocations, point mutations, and other clinical diseases.
REF: 228 OBJ: Level 1: Recall
5. 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.
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.
REF: 228 OBJ: Level 2: Interpretation
6. 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
C
The stability of a given hybrid can be calculated by determining the melting temperature of a probe. The melting temperature is the temperature at which 50% of the hybrids have formed and 50% of the single-stranded nucleic acid molecules are still dissociated. The melting temperature is dependent on the nucleotide composition of the probe.
REF: 228 OBJ: Level 1: Recall
7. 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.
B
Higher probe concentrations typically lower the reaction time by saturating all of the available probe target sequences. However, excessive probe concentrations promote non-specific binding of the probe to non-target sequences.
REF: 228 OBJ: Level 2: Interpretation
8. 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.
C
Many hybridization assay conditions are based on the expectation that a probe has exact or near-exact complementarity to the target nucleic acid. This will not always be the case. When conditions are stringent, exact matches between the probe and target will hybridize first, whereas mismatches form duplexes more slowly.
REF: 228 OBJ: Level 1: Recall
9. In a hybridization reaction, the pH affects the:
a. probe’s nucleic acids so that they do not lose their hydrogen atoms.
b. breakdown of the oligonucleotide pieces.
c. ability of the mismatches between the probe and the target to duplex.
d. stability of double-stranded nucleic acid molecules in solution.
D
pH affects the stability of double-stranded nucleic acid molecules in solution. An alkaline pH promotes dissociation of double-stranded molecules, whereas acidic pH solutions can depurinate probes and target nucleic acid molecules.
REF: 228 OBJ: Level 2: Interpretation
10. 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.
A
The function of the probe is to form a duplex with every complementary sequence available in the reaction, and the probe must be suited for the particular hybridization reaction that will be used.
REF: 228 OBJ: Level 1: Recall
11. Which of the following compounds are used for labeling probes?
a. Agar
b. Fluorescein
c. Digitoxin
d. Horseradish peroxidase
B
Nonisotopic labels include biotin, digoxigenin (DIG), and fluorescein.
REF: 228 OBJ: Level 1: Recall
12. 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.
C
In solid support hybridization (a technique often called “blotting”), the target nucleic acid is transferred and immobilized to a membrane, composed of either nitrocellulose or nylon. The solid membrane is often pretreated to reduce nonspecific probe binding to the membrane itself. Labeled probe is then hybridized to the immobilized nucleic acid, and washing steps are utilized to remove excess probe and clarify the signal.
REF: 229 OBJ: Level 2: Interpretation
13. The Southern blot test separates:
a. RNA.
b. proteins.
c. lipids.
d. DNA.
D
The Southern blot was first described in 1975 by E.M. Southern; he described a technique whereby chromosomal DNA is separated.
REF: 229 OBJ: Level 1: Recall
14. 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.
A
The DNA is first digested because chromosomal DNA is too large to separate in an agarose gel. Once the DNA has been separated and immobilized onto a solid membrane, labeled probe is hybridized to the specific target DNA sequence and detected.
REF: 229 OBJ: Level 1: Recall
15. A Northern blot is used to detect:
a. proteins.
b. RNA.
c. DNA.
d. lipids.
B
A Northern blot is used to detect RNA molecules.
REF: 229 OBJ: Level 1: Recall
16. 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.
C
Northern blots may be used to determine the size of particular RNA molecules and to semiquantitate the amount of a particular RNA transcript. The actual procedure of the Northern blot is similar to that of the Southern blot; RNA is separated in an agarose gel, transferred to a membrane, immobilized, and detected with a probe that hybridizes to the RNA species of interest. Detection is also accomplished to the type of label on the probe.
REF: 229 OBJ: Level 1: Recall
17. 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.
D
In situ hybridization, first described in 1969 by Pardue and Gall, is also not often used in clinical microbiology laboratories. This is a method of hybridization where DNA or RNA transcript can be detected directly in tissue with labeled probes. This technique is often performed directly in tissue that has been embedded in paraffin. In situ hybridization can also detect nucleic acids in intact cells and chromosomal genetic material.
REF: 230 OBJ: Level 1: Recall
18. 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.
A
Several manufacturers have developed useful assays that promote hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells. Generally, detection methods for these commercial systems are chemiluminescent based.
REF: 230 OBJ: Level 1: Recall
19. The Gen-Probe AccuProbe system which 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.
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 luminometer is used to detect these labeled duplexes. The luminometer 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.
REF: 230 OBJ: Level 1: Recall
20. 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
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.
REF: 231 OBJ: Level 1: Recall
21. Polymerase chain reaction (PCR) includes all the following steps except:
a. denaturation.
b. primer annealing.
c. primer extension.
d. gene expression.
D
Gene expression is the activation of DNA, which is not part of PCR. One PCR cycle consists of three steps: (1) denaturation of target DNA, (2) primer annealing to the target sequence, and (3) primer extension.
REF: 231 OBJ: Level 1: Recall
22. PCR requires all of the following components except:
a. DNA helicase.
b. DNA polymerase.
c. oligonucleotides (primers).
d. deoxynucleotide triphosphates.
A
Polymerase chain reaction (PCR) requires several components, including an enzyme commonly called DNA polymerase, a buffer for the polymerase, primers, the four deoxynucleotide triphosphates, and a source of template DNA (the target). DNA helicase is not required for this method.
REF: 231 OBJ: Level 1: Recall
23. What 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
B
An instrument called a thermal cycler is required. The instrument carries out the following “cycling”: target DNA is denatured at temperatures greater than 90° C, primers attach at 30° C, then amplification took place.
REF: 231 OBJ: Level 1: Recall
24. In a 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.
C
By denaturing the DNA, attaching the primers to the single-stranded DNA, then extending the primers to form duplicates of the target DNA, an exponential number of DNA copies can be made.
REF: 231 OBJ: Level 1: Recall
25. 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.
D
Because single-stranded DNA targets are necessary for PCR assays, the target double-stranded DNA is denatured or separated into single strands by raising the temperature to above 90° C.
REF: 231 OBJ: Level 1: Recall
26. 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.
A
The goal of this step is to hybridize, or anneal, oligonucleotide primers to the denatured, single-stranded target DNA strands. A pair of primers is used in standard polymerase chain reaction (PCR): one for each strand of dsDNA. The 5' ends of these primers frame the amplification region of the target DNA. Thus, the 5' ends of the primer pair define the eventual size of the PCR product. Primer annealing temperatures usually range from about 45° C to about 65° C for about 30 seconds to 2 minutes.
REF: 231 OBJ: Level 1: Recall
27. 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
C
The purpose of primer extension is to produce the PCR products. The DNA polymerase takes the individual nucleotides and adds them to the 3' end of each primer that is annealed to the target DNA strands. The target DNA strand acts as a reference strand for the polymerase.
REF: 232 OBJ: Level 1: Recall
28. The most commonly used polymerase is _____ polymerase.
a. Escherichia coli 37 DNA
b. Taq DNA
c. Mg DNA
d. Dds DNA
B
The Taq DNA polymerase was described first for use in polymerase chain reaction (PCR) and is still most commonly used.
REF: 233 OBJ: Level 1: Recall
29. What cation is required for the proper function of the Taq DNA polymerase?
a. Ca
b. Na
c. K
d. Mg
D
A divalent cation, usually Mg in the form of MgCl2, is required for the proper function of Taq DNA polymerase. Many DNA polymerase buffers are supplied with 1.5 mM MgCl2.
REF: 233 OBJ: Level 1: Recall
30. 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.
A
Because PCR is so sensitive, small amounts of extracted nucleic acid or carryover amplicons from previous PCR assays can contaminate future PCR assays. This may result in false-positive results. When contamination occurs, all equipment and work surfaces must be thoroughly cleaned, and new reagents (including primers) usually must be used. Laboratories that perform PCR and other amplification methods should, if possible, use separate rooms for template extraction, PCR reagent preparation, and amplification.
REF: 234 OBJ: Level 1: Recall
31. 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
B
The PCR amplicon is produced normally, but with uracil substituted for thymine. The enzyme uracil-N-glycosylase prevents replication of uracil-containing DNA. PCR reagents can be preincubated with this enzyme to ensure that amplicon carryover does not contaminate the reagents.
REF: 234 OBJ: Level 1: Recall
32. 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.
C
Ethidium bromide binds to nucleic acids by intercalating between bases. When ethidium bromide is irradiated with ultraviolet (UV) light, it fluoresces bright orange. This fluorescence can be visualized with the naked eye by holding a UV lamp over the gel, or with an imaging instrument that shines UV light up through the bottom of the gel.
REF: 235 OBJ: Level 1: Recall
33. 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.
D
Another major benefit to real-time PCR is that the reactions occur in closed tubes that do not have to be opened for detection. Thus there is a much smaller chance that amplicon from a real-time PCR assay will contaminate equipment, reagents, and work spaces.
REF: 236 OBJ: Level 2: Interpretation
34. What is the fluorescence resonance energy transfer (FRET) methodology?
a. The transfer of energy is made 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.
A
FRET is the transfer of energy from a donor dye molecule to an acceptor dye molecule; or FRET also occurs between a fluorescent dye and a quenching molecule that keeps emitted light low until the fluorescent dye is released from the quencher. For FRET to occur, the two molecules must be in close proximity to each other—they should be within about 1 to 5 nucleotides of each other.
REF: 236 OBJ: Level 1: Recall
35. 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.
B
Fluorescence from the reporter dye is kept to a low background level because of the close proximity of the quencher dye by fluorescence resonance energy transfer (FRET), where the fluorophore donates energy to the quencher dye. During the primer extension, 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, and then the entire probe is released. Fluorescence increases as the fluorophore is removed from the immediate vicinity of the quencher dye.
REF: 238 OBJ: Level 1: Recall
36. 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.
C
A Scorpion primer uses a single oligonucleotide to prime a specific sequence and to detect accumulation of PCR product.
REF: 240 OBJ: Level 1: Recall
37. 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.
D
The most sensitive technique available for detecting and quantifying messenger RNA is reverse-transcription PCR. The method uses an enzyme called reverse transcriptase to synthesize a complementary strand of DNA from an RNA template. The resulting cDNA is then used as a template in a PCR assay.
REF: 241 OBJ: Level 1: Recall
38. 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.
A
Some laboratories will use multiplex PCR to detect simultaneously two or more different targets from one PCR tube. This technique utilizes two different primer sets and is often used to detect an internal control in the same tube as the target of interest.
REF: 242 OBJ: Level 1: Recall
39. 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.
B
The assay itself basically serves as a form of internal control and ensures specificity. Nested PCR consists of two different, consecutive PCR assays. The first reaction is a standard PCR assay using one set of PCR primers. The amplicon produced from this first reaction is then used as the target in a subsequent PCR assay. The second primer pair is complementary to an internal region of the amplicon derived from the first PCR assay. Amplicon will only be synthesized during this second PCR assay if amplicon was produced in the first reaction.
REF: 242 OBJ: Level 1: Recall
40. 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.
C
NASBA stands for nucleic acid sequenced-based amplification.
REF: 242 OBJ: Level 1: Recall
41. 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.
D
The increase in nosocomial infection is a small-scale, limited problem. Most hospitals are required to tract their nosocomial infection rate. If the rate rises, all accrediting agencies indicate that facilities must use quality management and benchmarking to reduce the nosocomial infection rate. The other three choices constitute global problems, which require a global solution.
REF: 246 OBJ: Level 1: Recall
42. 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.
A
Nonamplified typing techniques include Southern blotting, plasmid profile analysis, restriction enzyme analysis of chromosomal DNA, pulsed field-gel electrophoresis, and multilocus enzyme electrophoresis.
REF: 246 OBJ: Level 1: Recall
43. 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.
B
Amplified methods that are commonly used include arbitrarily primed PCR, also called random amplified polymorphic DNA, repetitive palindromic extragenic elements PCR, and multilocus sequence typing.
REF: 246 OBJ: Level 1: Recall
44. 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.
C
This technique is similar to the Southern blot. Chromosomal DNA is extracted from isolates of interest and digested with a restriction enzyme. The enzyme used for digestion should cut the chromosomal DNA in several places, so that small and large fragments are produced. The resulting RFLP pattern is analyzed by agarose gel electrophoresis; transfer to a membrane and subsequent use of a probe to identify a specific sequence is not performed.
REF: 246 OBJ: Level 1: Recall
45. What 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)
D
A restriction enzyme is used to digest chromosomal DNA at a small number of sites along the chromosome. This results in large DNA fragments that are difficult to resolve by standard agarose gel electrophoresis. During PFGE, these large fragments are separated in a low-percentage, low-melt agarose gel by an angled electrical field that periodically changes orientation. These changes in electrical field orientation are pulses that successfully “force” the large DNA fragments through the agarose gel and separate them by size. A low-percentage agarose gel is used for the large DNA fragments, and low-melt agarose also enables large fragments to migrate easier.
REF: 247 OBJ: Level 1: Recall
46. What 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
A
Multilocus enzyme electrophoresis involves the extraction of proteins from isolates of interest, followed by electrophoretic separation and selective staining of these proteins. The expression of the protein’s genotype is reflected in the position of the stained band, according to the protein’s mobility. Mobility is determined by the net charge of the protein and also by the structure of the protein. If there are two bands of the same protein in different positions after separation, then this suggests two different conformations of the protein—two alleles of the same gene.
REF: 247 OBJ: Level 1: Recall
47. What 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
B
Random amplified polymorphic DNA is a popular method of DNA fingerprinting. Small primers with random sequences are used during this technique. These primers do not have a specific target. Instead, the random primers indiscriminately amplify chromosomal DNA during polymerase chain reaction (PCR) cycles. This results in fragments of varying lengths after separation by agarose gel electrophoresis. A particular strain will have a different fragmentation pattern than other strains.
REF: 247 OBJ: Level 1: Recall
48. 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.
C
The information obtained from sequencing an organism’s genome allows for comprehension of cellular processes, protein associations, and interrelatedness of biologic activities.
REF: 248 OBJ: Level 1: Recall
49. 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
D
The term DNA microarray refers to a microscopic grouping of DNA molecules attached to a solid support mechanism. Silicon chips, glass, or plastic have been used as the solid surfaces to support an array. DNA microarray is sometimes referred to as a DNA chip, or a gene chip. A DNA microarray gives investigators the potential to evaluate gene expression from an entire organism, or even from several organisms. DNA microarrays are often used to analyze transcriptional levels of genes during a particular disease state.
REF: 250 OBJ: Level 2: Interpretation
50. 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
A
Genomic sequencing has also led to an increased understanding of protein interrelationships and expression in cells. Proteomics is the study of proteins on a cellular level. Like genomics, proteomics is a large-scale process, but it is probably more complicated than analysis at the gene and transcriptional levels.
REF: 250 OBJ: Level 1: Recall
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