Chapter 9 - Molecular Diagnostics
In a chain-termination DNA sequencing reaction, which one of the following components are tagged with a fluorescent dye? a. Dideoxynuclotide triphosphates (ddNTP) b. Deoxynucleotide triphosphates (dNTP) c. Pyrophosphates (PPi) d. Capillary probe (CaP)
a. After the generation of terminated fragments in the cycle sequencing steps, fragments are tagged with a fluorescent dye labeled terminator ddNTPs then separated by denaturing polyacrylamide gel or capillary electrophoresis.
The HLA A, B, and C molecules are encoded for by which class gene(s) within the major histocompatibility complex (MHC)? a. Class I b. Class II c. Class III d. All groups in MHC class
a. Genes encoding the class I molecules, which includes HLA A, B, and C are considered class I genes. Class II consists of DR, DQ, and DP.
The field of proteomics studies which of the following? a. Proteins on a cellular level b. Serum proteins c. Proteins in genes d. The human genome
a. Genomic sequencing has 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.
In a real-time PCR assay, which probe type is used in conjunction with fluorescence resonance energy transfer (FRET) on the formation of a duplex? a. Hybridization b. Hydrolysis c. Hexamere d. Primer
a. Hybridization probes are those that reversibly change fluorescence on duplex formation. Fluorescence resonance energy transfer techniques depend on the proximity between two distinct fluorescent labels. When the two labels are brought closer together through hybridization, fluorescence is released.
The detection of the DNA from cytomegalovirus (CMV) and human papillomavirus (HPV) is typically performed using the Hybrid Capture assay. What type of assay is Hybrid Capture? a. Target amplification assay b. Signal amplification assay c. Reverse transcriptase assay d. Viral load assay
b. Hybrid capture is a signal-amplification assay that detects cytomegalovirus (CMV) and human papillomavirus (HPV) DNA in specimens. Hybrid capture methods use a bound antibody that is specific for RNA-DNA hybrid molecules that are formed during solution-phase hybridization of a DNA sample and an unlabeled RNA probe. The signal from the complex is amplified and detected.
During the replication process the addition of bases occurs: a. At the telomeric end of a DNA strand b. In the 30 to 50 direction c. In the 50 to 30 direction d. In both the 50 to 30 and 30 to 50 directions
c. DNA polymerase III synthesizes a daughter strand in the 50 to 30 direction only, because nucleotides are only able to be added to the 30 carbon end of the sugar.
When RNA is to be used in a PCR amplification procedure, what is the first step that must be performed? a. RNA cannot be used in a PCR reaction because it will disintegrate during the denaturation step b. RNA must be denatured to form single strands to allow for the annealing of primers c. A reverse-transcription procedure must be performed to form cDNA d. RNA must first be treated with RNases to remove interfering substances from the target
c. RNA targets can be amplified into DNA sequences when they are initially transcribed into cDNA using reverse transcription. Some thermostable enzymes have both DNA polymerase and reverse transcription activities, so both steps can be performed in the same tube with the same enzyme.
Solid-phase DNA extraction procedures are more commonly used in a clinical laboratory because: a. They can be coupled with a gas chromatograph linked to a mass spectrometer b. They perform best when a large volume of sample is submitted for DNA extraction c. They are adaptable to automation d. They involve organic solvents that are easily found in a laboratory
c. Solid-phase extraction methods are amiable for high-throughput and adaptable for automated processing.
Which of the following is not a factor that influences hybridization reactions? a. Degree of complementarity between the probe and target nucleic acid b. pH c. Size of the target's genome d. Temperature
c. The size of the target's genome does not affect hybridization, but the target nucleic acid sequence does, along with pH and temperature.
Which one of the following statements concerning mitochondrial DNA (mtDNA) is incorrect? a. Pseudogenes are small pieces of nuclear DNA that share significant homology with mtDNA b. mtDNA is circular and contains approximately 16,500 base pairs c. mtDNA is inherited from the mother because only ova contain mitochondria d. Follows mendelian inheritance patterns
d. Mitochondrial DNA does not follow the Mendelian inheritance pattern.
Which of the following genes is not found in retroviruses? a. Gag b. Pol c. Env d. Onc
d. The typical retroviral genome contains at least three genes: gag, pol, and env.
The technique that uses fluorescent DNA probes to detect chromosomal abnormalities within cells in cytogenetic studies is referred to as: a. Fluorescence in situ hybridization (FISH) b. Karyotype in situ hybridization (KISH) c. Fluorescence in situ PCR d. Microarray
a. In a fluorescence in situ hybridization (FISH) assay, a fluorescent DNA probe is hybridized to a specific locus to produce a colored signal. A translocation would appear as the color produced from the combination of two different fluorescently labeled probes.
Which of the following is false about primers? a. Primers should be at least 100 nucleotides long b. Primers are typically 15 to 30 nucleotides long c. Primers should have a GC percentage of 40% to 60% d. Primers should anneal to a specific target
a. Primers should not be larger than 30 base pairs long. Primer design should take into account melting temperature (Tm) for both the forward and reverse primers.
How does ribonucleic acid (RNA) differ from deoxyribonucleic acid (DNA)? a. RNA has a uracil and DNA has a thymine b. RNA does not contain nucleotides and DNA does c. DNA resides in the cytoplasm of the cell and RNA is in the nucleus d. DNA has a messenger form and RNA does not
a. RNA differs from DNA in that it has uracil that replaces thymine. RNA is also single stranded.
Identify the correct sequence of events for a polymerase chain reaction (PCR) cycle. a. Anneal, extend, and denature b. Denature, anneal, and extend c. Extend, anneal, and denature d. Extend, denature, and anneal
b. Denaturing the target duplexes into single strands is the first step in polymerase chain reaction (PCR). Next, the sample is allowed to cool and the primers anneal specifically to the complementary sequences on the target. The primers are extended by the polymerase.
The differential activation of genes depending on the parent from which they are inherited is referred to as: a. Allelic heterogeneity b. Imprinting c. Mosaicism d. Pleiotropy
b. Imprinting refers to the differential marking of specific paternally and maternally inherited alleles during gametogenesis, resulting in differential expression of those genes.
The major advantage for using nucleic acid techniques for the identification of infectious disease is: a. The lower cost involved with molecular analysis b. The high specificity for identification of an organism c. The lack of false negatives d. The ability to distinguish normal flora from disease-causing organisms
b. Molecular techniques are designed to achieve high specificity for the organism that the test is designed to detect; however, the techniques are subject to false-negative results because of the presence of inhibitors. Additionally, detection of nucleic acid of a pathogen does not ensure that the organism is the cause of the disease. The organism might be forming part of the normal flora, colonizing a specific area, or causing infection but not disease.
What is the preferred specimen type for molecular studies for the diagnosis of inherited mutations? a. RNA extracted from peripheral mature red cells b. RNA extracted from fresh serum c. DNA extracted from peripheral blood white cells d. DNA extracted from peripheral blood mature red cells
c. DNA extracted from peripheral blood (or bone marrow) white cells is the preferred specimen for molecular techniques when they are used to diagnose inherited disorders. In inherited disorders, the DNA from any cell could theoretically be used because the DNA at the molecular level will be identical regardless of cell origin. However, DNA from peripheral blood white cells is the easiest tissue to obtain. Mature red blood cells cannot be used because they do not have a nucleus and therefore do not have any DNA.
A type of polymorphism that consists of a series of trinucleotide repeats that can be two to seven base pairs in length and is known as a microsatellite sequence is also referred to as a: a. Restriction fragment length polymorphism (RFLP) b. Variable number tandem repeat (VNTR) c. Restriction endonuclease d. Short tandem repeat (STR)
d. A short tandem repeat (STR) consists of DNA sequences that have a core sequence of two to seven base pairs, which are repeated in tandem several times. An example of a trinucleotide repeat occurring four times is 5' GTG-GTG-GTG-GTG 3'.
Which of the following is a block of specific sequence variants that are inherited together? a. Allele b. Haplotype c. Locus d. Polymorphism
b. Alleles linked when inherited are called haplotypes. The location of a gene on a chromosome is the locus, and the possible alternative forms of the gene at the locus are referred to as alleles. The variations in alleles are known as polymorphisms.
Transfer RNA (tRNA): a. Is a gene silencing RNA used in cancer research b. Is a macromolecular complex delivered from the nucleus by ribosomal RNA c. Contains the codon sequence that synthesizes an amino acid d. Contains the anticodon region that binds to mRNA codon in the ribosome
d. mRNA with its three-nucleotide codon will bind to the tRNA molecule's anticodone, which then carries the attached amino acid to a ribosome to be added to a growing peptide chain.
This method is based on the microscopic grouping of probe DNA molecules attached to a solid support mechanism such as glass, silicon, or plastic chips. a. DNA microarray b. Multilocus enzymes electrophoresis c. Restriction fragment length polymorphism (RFLP) d. Polymerase chain reaction (PCR)
a. The term DNA microarray refers to a microscopic grouping of DNA molecules attached to a solid support mechanism. A DNA microarray is also referred to as a DNA chip or gene chip. A DNA microarray allows for the detection of gene expression or possible deletions using comparative genome hybridization (CGH).
The site of a particular nucleotide sequence on a chromosome is referred to as a(n): a. Allele b. Locus c. Polymorphism d. Genotype
b. A locus is a location of a gene where polymorphic alleles are found. The alleles at a locus are the genotype for the locus.
The enzyme ligase joins the Okazaki fragments of the __________________. a. Template strand b. Lagging strand c. Leading strand d. Primer fragments
b. The lagging strands are discontinuously assembled and require ligase to join the fragments.
Messenger RNA (mRNA) is produced in the _____________. a. Golgi b. Mitochondria c. Nucleus d. Ribosomes
c. Messenger RNA (mRNA) is produced in the nucleus of the cell. After processing, the mature mRNA leaves the nucleus and enters the cytoplasm, where it is translated.
The rate of DNA migration in a gel electrophoresis depends primarily on the ____________. a. Buffer temperature b. Pore size of the gel c. Shape of the DNA molecule d. Size of the genomic DNA
d. The sizes/molecular weight of the genomic DNA fragment can be estimated by gel electrophoresis using size standards.
In chronic myeloid leukemia (CML) the fusion of chromosomes 9 and 22 produces a hybrid gene, BCR-ABL. This chromosome is referred to as the: a. Cincinnati chromosome b. Legionnaires chromosome c. Myeloprolifererative chromosome d. Philadelphia chromosome
d. The translocation involving chromosomes 9 and 22 known as the Philadelphia chromosome is a hallmark in the diagnosis of chronic myelogenous leukemia (CML). The resulting protein has enhanced tyrosine kinase activity. t(9:22) may be seen in acute lymphoblastic leukemia (ALL) with a poor prognosis.
The purity of an extract of nucleic acid can be determined by which of the following? a. Measuring absorbances at 260 nm and 280 nm and dividing A260 by A280 b. Measuring the bands on an agarose gel c. Measuring absorbances at 260 nm and 280 nm and subtracting A280 from A260 and then multiplying the result by a dilution factor d. Multiplying the concentration of the nucleic acid by the dilution factor
a. Purity of extracted RNA and DNA can be evaluated by assessing the ratio of the absorbances at 260 nm and 280 nm (A260/A280). A ratio of 2.0 indicates a pure DNA extract.
The chromosomes in a eukaryotic cell: a. Are in their most compact state and appear as chromatin arms joined at the center during the cell division stage called metaphase b. Contain genomic regions that are rich in genes, less compactly organized, and termed heterochromatin c. Contain two specialized regions of euchromatin, telomeres, and centromeres d. Are highly ordered structures of a single RNA molecule, compacted many times with the aid of structural RNA-binding proteins
a. Response a is the only correct answer; b is incorrect because the gene-rich regions are euchromatin; answer c is incorrect because telomeres and centromeres are heterochromatin; d is incorrect because chromosomes are composed of DNA.
Signal amplification differs from target amplification when designing protocols for identification of nucleic acids. Which of the following is an example of a signal amplification technique? a. Branched-chain DNA detection b. Ligase chain reaction c. Polymerase chain reaction d. Reverse-transcriptase PCR
a. Signal amplification techniques use nucleic acids to increase the signal for detection. The branched-chain DNA (bDNA) method is one of these techniques.
An exon is defined as: a. A region of DNA present in a mature strand of mRNA and can be translated into protein b. A region of DNA that is recognized by RNA polymerase to start transcription c. A region of DNA that is transcribed then removed from mRNA by excision and is not translated into protein d. A region of DNA comprising three base pairs that signal for termination of replication
a. The coding sequence in a gene is divided into regions called exons. These coding regions specify the amino acid sequence in the resulting protein. Introns are the noncoding regions of a gene. The promoter region regulates initiation and rate of transcription.
When performing a PCR procedure, which is the most important control to run to check for the presence of amplicons? a. Blank control b. dTTP control c. Control d. Oligo dT control
a. The negative control or blank (all reactants minus target DNA) is one of the most important controls when providing quality control for a polymerase chain reaction (PCR) run. If the blank demonstrates DNA bands, the master mix has likely been contaminated with PCR reaction product from a previous run.
Which of the following are forms of nucleic acids? a. Nucleosides b. DNA or RNA c. Base pairs d. Trinucleotide sequences
b. Nucleic acids include a sugar moiety (2-deoxyribose in the case of DNA), a phosphoric acid, and purine or pyrimidine base; deoxyribonucleic acid is DNA and ribonucleic acid is RNA. Nucleosides comprise a base and a sugar.
Which term describes a normally occurring gene that when altered is often associated with cancers? a. Oncogene b. Proto-oncogene c. Meta-oncogene d. Post-oncogene
b. Oncogenes are derived from protooncogenes, which are normal cellular genes. The protooncogenes become oncogenic and are often associated with cancers when they have been altered by dominant mutations.
PCR requires all of the following components except: a. Deoxynucleotide triphosphates b. DNA endonuclease c. DNA polymerase d. Oligonucleotides (primers)
b. Polymerase chain reaction (PCR) requires several components: DNA polymerase, a buffer for the polymerase, primers, the four deoxynucleotide triphosphates, and a source of template DNA (the target). DNA endonuclease is not required for this method.
During replication the "parent" strand of DNA serves which purpose? a. It is completely excised by exonuclease enzymes when replication of the strand is complete b. It has a sequence that is complementary to the daughter strand that is being replicated c. It is also referred to as the lagging strand d. It will be copied by a DNA polymerase to form two new daughter DNA strands
b. The sequence of a single strand of DNA gives rise to the sequence of its complementary strand. During replication the complementary strands are the "daughter" strand. Replication is semiconservative, meaning the new dsDNA is made up of a parent and daughter strand.
The most common nucleic acid stain used after separation by agarose gel electrophoresis is: a. Bromothymol blue b. Bromocresol 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. This fluorescence can be detected by holding a UV lamp next to the gel or using visualization software.
In which of the following inheritance patterns are homozygous alleles necessary to express the disease phenotype? a. Autosomal dominant b. X-linked dominant c. Autosomal recessive d. Trinucleotide repeats
c. In autosomal recessive disease, two abnormal alleles at a given locus (by receiving one mutant allele from each carrier parent) are necessary to produce the disease phenotype. Affected individuals may be homozygous with two copies of the same mutation.
In the organic liquid phase (phenol-chloroform) DNA extraction procedure, proteins are precipitated out of solution: a. In the aqueous phase b. As a pellet on the filter c. In the organic phase d. On the silica-based gel
c. In the organic extraction method, cellular debris and protein are separated by organic solvent extraction from the hydrophilic soluble DNA fraction. The DNA will be in the aqueous phase of the biphasic solution.
Which enzyme catalyzes DNA replication? a. Endonuclease b. Ligase c. Polymerase d. Reverse transcriptase
c. Polymerases catalyze the synthesis of complementary nucleic acid polymers using a parent strand as a template. In vitro, these enzymes can extend an oligonucleotide primer that is annealed to a template strand.
The process of transferring the digested DNA out of a gel after electrophoresis and onto a nylon membrane is referred to as: a. Hybridization blotting b. Northern blotting c. Southern blotting d. Western blotting
c. Southern blotting is the classic method for detecting large segments of DNA that are not easily amplified. The original sample DNA is digested by a restriction enzyme, separated by electrophoresis, and transferred to a solid support, followed by selective visualization of fragments by hybridization of labeled probes.
The central dogma speaks to the function of the molecular components of DNA and states that: a. The main function of genes is to store and transmit genetic information b. There are specific nucleotide triplets that code for specific amino acids c. Genes are perpetuated as sequences of nucleic acid, but function by being expressed in the form of protein d. All sequences of DNA in the human genome will result in the production of RNAs
c. The "central dogma" proposes that DNA replicates from DNA, DNA is transcribed to RNA, and RNA is translated to produce proteins. Only genes (not all DNA sequences) are expressed as proteins, and specific sequences of nucleic acid make up a gene.
Which of the following statements is true regarding agarose gel electrophoresis? a. Nucleic acids are separated in an electrical field because of their net positive charge b. Larger nucleic acid molecules are able to migrate through the agarose gel faster than smaller molecules c. Nucleic acids are separated in agarose by shape, charge, and size d. Agarose is a dye that binds in double-stranded DNA
c. The migration of a nucleic acid fragment through agarose gel depends on the size, shape, and charge of the fragment.
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 amplicons d. To activate the DNA polymerase to form hybrids
c. The purpose of primer extension is to produce amplicons, which are the resulting fragments from a polymerase chain reaction (PCR) cycle. The DNA polymerase takes the individual nucleotides and adds them to the 30 end on each primer that has annealed to the target DNA strands. The target DNA strands act as the reference for the polymerase.
Pyrosequencing sequence analysis and quantification depends on the release of ________ in a quantity equal to that of an incorporated dNTP. a. Apyrase b. Luciferase c. Nucleotides d. Pyrophosphate
d. In pyrosequencing, each incorporation event is accompanied by release of a pyrophosphate (PPi), so the quantity of PPi produced is equimolar to the amount of incorporated nucleotide. The conversion of PPi and adenosine 50 phosphosulfate into adenosine triphosphate (ATP) by the ATP sulfurylase allows ATP to drive the conversion of luciferin into oxyluciferin, which generates visible light. The light produced is proportional to the number of nucleotides incorporated.
In the PCR, a _____________ initiates extension of the sequence of interest by allowing Taq polymerase to begin adding nucleotides to single-stranded DNA. a. Probe b. Ligase c. Promoter d. Primer
d. Primers are provided in great excess and specifically anneal to a complementary sequence on the target DNA. Once the primers have annealed, the action of the polymerase synthesizes two additional DNA strands containing the primers as the 50 ends.
The increase in quantifiable signal observed early in a real-time PCR run depends on which of the following? a. The wavelength of the fluorescent dye in the reaction b. The number of cycles in the run c. The amount of fluorescent quencher d. The initial amount of target DNA
d. Real-time polymerase chain reaction (PCR) monitors the amount of product formed each cycle by systematically quantifying the fluorescence signal. The fluorescent signals depend on the amount of target DNA present in the sample.
Which of the following is a description of restriction endonucleases? a. A family of bacteria from which endonuclease that cuts DNA into fragments is produced b. Only able to digest specific genes on specific chromosomes with their endonuclease action c. Enzymes that specifically degrade DNA in nucleic acid mixtures when plasmids are present d. Enzymes produced by bacteria to prevent invasion and replication of foreign DNA in their bacterial genome
d. Restriction enzymes are sequence-specific endonucleases that cut dsDNA into fragments, resulting in either blunt-ended or sticky-ended pieces. These endonucleases are isolated from specific bacteria that use the enzymes as protection from invasion by foreign DNA.
A tumor-suppressor gene performs which of the following task(s)? a. Codes for normal growth-promoting proteins b. Codes for proteins that control cell division c. Repairs nucleotide mismatches in the DNA strand d. All of the above
d. The proteins encoded by many tumor-suppressor genes are involved in the regulation of progression through the cell division cycle or in the regulation of DNA repair.
Which of the following practices should be employed to prevent contamination of patient samples and reagents with amplicons? a. Use bleach solution for cleaning work area b. Use UV lights in hooded work area c. Maintain closed analytical systems d. All of the above
d. Ultraviolet-induced cross-linking of DNA can minimize the effects of amplicon contamination during sample setup; laboratory surfaces should be regularly cleaned using amplicon-decontaminating agents, such as solutions of bleach, and closed analytical systems (i.e., reaction tubes that are analyzed without being opened) should be used to reduce contamination with amplicon.
