™ 6: Molecular Biology | Molecular Science Questions | Molecular Biology ‘Tre following items have been identified generally as appropriate for bath entry level medica laboratory scientists and medical laboratory technicians Items that are appropriate for medical laboratory scientists only re marked with an “MLS ONLY” | 369 Questions 377 Answers with Explanations | ‘369 Molecular Science ‘978 Molecular Science 1370 Molecular Techniques 378 Molecular Techniques 374 Applications of Molecular Testing 379 Applications of Molecular Testing Molecular Science || 1. Which nucleotide pair has a high frequency of mutation in human chromosomal DNA? Be cc 2 During the G2 phase of the cell cycle, the DNA content ofa cells: haploid & diploid || € triploid @ tetraploid 3 Intervening sequences are found in 12 heteronuclear RNA b mature mRNA © ribosomal RNA 4 transfer RNA 4 Which is considered an epigenetic modification of DNA? fa transversion of an A nucleotide toa T in an exon of a gene b atransition of an A to.aG in an intron of a gene methylation of CpG islands in the promoter region of a gene 4. insertion of a nucleotide into the coding region of a gene 5A metaphase chromosome with primary constriction that gives the chromosome clearly defined short and long arms is considered: a acrocentric b submetacentric © metacentric 4 telocentric Reverse transcriptase would best be described asa a DNA-dependent DNA polymerase | | gee DNA-dependent RNA polymerase | RNA-dependent DNA polymerase RNA-dependent RNA polymerase aoge | cl Laboratory Certification Beaminations 369 |Molecular Biology | Molecular Techniques Questions 7 The 2alleles for a sex-linked recessive disease are X and x. The mutant allele s x. What is the percentage of male offspring that would be expected to be affected by the disease from parents who have the following genotypes? mother: Xx father: XY a0 b 25 © 50 a 100 8 The term that best describes males regarding X-linked genes is 4 heterozygous b homozygous ¢ haplozygous 4d hemizygous 9 The mode of inheritance of mitochondrial DNA is: dominant recessive codominant maternal aoce Molecular Techniques 10 Which statement concerning the resolution of DNA on an agarose gel is true? SM a. the polymerization of agarose is initiated by adding TEMED and ammonium persulfate to ‘agarose monomer in solution 'b agarose gel electrophoresis can resolve smaller molecules of DNA compared to acrylamide gel electrophoresis, «€ as the concentration of agarose in the gel increases, the smaller the size of DNA that can be resolved on the gel d_ ONA must be denatured before being loaded into the sample wells of an agarose gel 11 An RFLP is most likely detected using which ofthe following methods? lv a Northern blot b Southern blot ¢ Western blot Southwestern blot 12. The methodology of performing a reverse dot blots best described as Mer g_ attaching many patient DNA samples to nitrocellulose membrane and hybridizing the patient samples with a single labeled probe bb attaching multiple patient samples to a nitrocellulose membrane and hybridizing multiple labeled probes to the patient samples «attaching multiple labeled probes t0 a nitrocellulose membrane and hybridizing a patient sample to the multiple labeled probes 4 attaching multiple probes to a nitrocellulose membrane and then hybridizing a single labeled patient sample to the multiple probes 13 Which condition has the highest stringency for washing a Southern blot after hybridization has Wily been completed? ‘8 low temperature, low salt concentration b high temperature, low salt concentration ‘€ high temperature, high salt concentration 4. low temperature, high salt concentration 370 The Board of Certification Study Guide6: Molecular Biology | Molecular Techniquee Questions 14 18 19 2 ‘The Klenow fragment of Ecoli DNA polymerase | is used: ‘a to make cDNA from an RNA template b tolabel DNA probes by random primer labeling ¢ in quantitative real time PCR 4 tolabel a DNA probe by nick translation What isa solution hybridization method for the detection of nucleic acid:protein interactions? ‘a RNase protection assay B gel mobility shift assay € strand displacement amplification assay hybrid capture assay Which probe is most often used to detect trisomy 21 by interphase FISH? locus-specific probe b alpha satellite DNA probe € chromosome paint probe spectral karyotyping probe How many volumes of ethanol are added to 1 volume of a DNA:salt solution to cause the DNA to precipitate? Chelex® 100 resin is used to 1 purify mRNA from total RNA extract total RNA from cells € extract DNA from cells 4 remove unincorporated primers from PCR reactions prior to gel analysis of PCR products ‘The fluorescent dye that exhibits the greatest sensitivity for quantization of DNA is: ethidium bromide b Hoechst 33258 © propidium iodide @ SYBR* Green ‘When quantifying the amount of genomic DNA in a sample by spectrophotometry, ti OD 260 of 1.0 corresponds to what concentration of DNA? 2 20u6/mL, b 20ng/mt © 50ug/mL 100 g/mL. ARNA sample is isolated from peripheral blood cells of a patient. When performing spectrophotometric analysis to determine the yield of RNA in the sample you find the 1:40 dilution of the 0.5 mL sample gives an OD 260 reading of 0.03125 and an OD 280 reading ‘of 0.01760. What is the total amount of RNA contained in the 0.5 ml. sample? ze (Clinical Laboratory Certification Beaminationt 3726: Molecular Biology | Molecular Techniques Questions 22 Which statement is true concerning the analysis of short tandem repeats (sto? OMY a str are detected by RT-PCR b stralleles are determined by Southern blotting € stralleles are determined by PCR product size 4. str analysis requires that high molecular weight genomic DNA be available in samples to be tested 23 Which reagent generates a signal during the annealing stage of a quantitative real time My PCR ceaction? a SYBR* Green b TagMan* probe € molecular beacon 4 Scorpion™ probe 24 Denaturation of DNA during a PCR reaction refers to breaking: 42 hydrogen bonds between nitrogenous bases in base-paired nucleotides b phosphodiester bonds between nitrogenous bases in base-paired nucleotides € covalent bonds between nitrogenous bases in base-paited nucleotides 4 peptide bonds between nitrogenous bases in base paired nucleotides 25. Whatis the most critical step in determining the specificity of a PCR reaction? 44 denaturation temperature annealing temperature extension temperature 4 number of cycles in the PCR reaction 26 APCR reaction in which 4 different sets of primers are used to simultaneously amplify 4 distinct Toci in the same reaction tube is known as 3: 4 multiplex PCR reaction b heteroplex PCR reaction « polyplex PCR reaction 4 quadraplex PCR reaction 27, Avatiation ofa standard PCR reaction that can increase the sensitivity and specifcty of a low {8 copy number target in a patient sample is known as 4 branched PCR b RT-PCR € nested PCR 4. cleavage-based PCR 28 Probes are often used during real time PCR to quantitate the formation of specific amplicons ‘May during the reaction, Which system involves the use of 2 distinct probes to generate a fluorescent signal? TagMan® b molecular beacon ¢ FRET 4 Scorpion™ 29 Which technique employs the amplification of the signal cesulting from probe:target {iy hybridization rather than by amplifying the target othe probe? ‘4 branched DNA analysis polymerase chain reaction « ligase chain reaction nucleic acid sequence-based amplification 372. The Board of Certification Study Guide6: Molecular Biology | Molecular Techniques Questions 30 Refer to the figure, An amplicon for gene X is 176 base pairs (bp) in size. A variant allele x, gives KS, the same size amplicon but has an Alwl restriction enzyme recognition site that is not present in the amplicon arising from allele X. A PCR reaction is run on a DNA sample isolated from 3 patients. The sample is digested with Alwl after the PCR is complete and products ofthe digestion are resolved on a 4.0% agarose gel (- = no Alwl digestion, + = Alw! digested), ‘Assuming all appropriate controls have worked correctly, what is the interpretation of the ‘genotype of patient #3 with regards toalleles X and x? & patient is homozygous for X b patient is heterozygous for X and x patient is homozygous for x 4 patient's genotype cannot be determined 31. Methylation of cytosine residues is involved in the alteration of gene expression in a number S83, of cancers. Which sequencing technique is best suited to determine if methylation of cytosine residues has taken place in the promoter region of a given gene? 2 Maxam/Gilbert chemical cleavage b Sanger dideoxyterminator € bisulfite sequencing 4 pyrosequencing 32 Which method is best suited to screen for mutations in @ gene having multiple Bay possible mutations? ‘& comparative genome hybridization b allele-specific PCR € realtime PCR single-strand conformational polymorphism 33 What is the purpose of the enzyme uracil-N-glycolase in a molecular diagnostics laboratory? Sg tolower the melting temperature of RNA:ONA hybrids in Northern blot analysis b toxemove any previously generated amplicons containing dUTP from PCR reactions € toremove contaminating genomic DNA from an RT-PCR reaction 4 tomelt double-stranded DNA to single-stranded DNA during a Southern blot procedure 34. What is the best method to rule out the possibilty of genomic DNA giving a false-positive result Biiy im an RT-PCR reaction? 1 use of upstream and downstream primers that span an exon-intron-exon region of the target B treatment of RIA that willbe used in the RT reaction with RNase € addition of uracil-N-glycolase to the RT reaction 4 omission of Tag DNA polymerase from the PCR reaction Clinical Laboratory Certification Examinations 373.6: Molecular Biology | Applications of Molecular Testing Questions Applications of Molecular Testing 35. Monitoring the load of HIV circulating in a patient's plasma would best be accomplished using. Efe which method? a PCR b DNA sequencing © NABSA 4 bDNA 36 A common use for pulsed-field gel electrophoresis is SRY a DNA fingerprinting mitochondrial DNA typing € epidemiological typing of bacterial strains 4 tumor cell phenotyping 37 When genotyping strains of bacteria by PEGE, what is the minimum number of genetic Unie differences that must be observed between a reference strain and a test strain before it can be determined that the test strain is unrelated to the reference strain? al b2 <3 aa 38 A human actin gene may be used as an amplification control when performing PCR testing for [Shiv Mycoplasma pneumoniae in a bronchoalveolar lavage taken from a human patient. The type of amplification control is described 3s: ‘2 homologous extrinsic homologous intrinsic heterologous extrinsic heterologous intrinsic 39° Molecular-based testing was performed on a nasopharyngeal swab sample taken from a patient Bil Results are reported as positive for [S481 and negative for 1S1000. These results indicate the patient's nasopharynx is colonized with: 18 Bordetella pertussis b Bordetella holmesii «Bordetella parapertussis 4 Bordetella sp 40. DNA-based testing for the presence of MRSA uses PCR with primers specific for which gene? Bee ena b inha mec a rpod 41 Atranslocation in which chromosome pair creates a BCR/ABLI fusion gene product associated with CML? a 1118 1418 © 922 4 914 ‘374 The Board of Certification Study Gulde6: Molecular Biology | Applications of Molecular Testing Questions 42 _ The molecular-based diagnostic test for Mycobacterium tuberculosis uses ribosomal RNA as ‘Shy. the target. Primers are made to be specific for which RNA subunit? a 5.88 b 16s € 188 4 285 43 APCR-based assay is performed to determine the clonality of B cells in a patient using a forward Sine primer specific for the innermost framework region and a reverse primer complimentary to the joining region (of the immunoglobulin heavy chain). When run on an agarose gel containing ‘ethidium bromide, the patient sample exhibits a smear of staining over a wide range in the patient sample lane along with one distinct band approximately a third of the way dawn from the sample loading well. Assurning all controls worked properly for the assay, what is your interpretation of, the results for this patient? 4 patient is normal patient has mononucleosis patient has a polyclonal population of B cells 4 patient has a moneclonal population of B cells 44 After performing interphase FISH with X chromosome-specific and Y chromosome-specific Mgy_ alpha satelite probes, it i determined that nucle: in cells from your phenotypic female exhibit only 1 bright Guorescent spot with the X chromosome-specific probe and do not react at all with the ¥-specific probe, Assuming all controls have worked correctly, what is the most likely iagnosis for this patient? ‘4 patient isa genetically normal female b patient isa female with Turner syndrome € patient isa fernale with fragile X syndrome 4 patient isa male who suffers from androgen insensitivity syndrome due to the lack of a chromosome 45. A Robertsonian translocation may be associated with which genetic disorder? "Sa Turner syndrome Bb Down syndrome ‘€ Angelman syndrome 4 Cridu chat syndrome 46 A molecular-based test commonly used in testing donors and recipients prior to organ {hy transplantations is 1 sequence-specific PCR bb allele-specific PCR eal time PCR 4 roverse transcriptase PCR Clinical Laboratory Certification Examinations 3756: Molecular Biology | Applications of Molecular Testing Questions 37 The results of st typing of a child, the child's mother and 3 alleged fathers (AF) of the child are MS, listed in the table below. Locus Child = Mother AF AFZ Ara ame genotype genotype genotype genotype genotype csFIPO 10,12 rate nme 1012 12,19 TPOX 99 an a an an TH 79 793 78 79 793 Frsaoy 6.9 a9 69 66 99 FESFPS 10,11 11.13 waa 710 109 WA 1418 16,18 1318134 15.18 Which of the statements below is correct concerning the alleged fathers? ‘a AFL is most likely the father of the child, b AF2 is most likely the father of the child ‘¢ AF3 is most likely the father of the child 4d None of the alleged fathers is likely the father of the child. 48. DNA sequencing of hypervariable regions I and Ili used to: RY a detect polymorphisms in human TPS3 1 genotype strains of Mycoplasma to monitor chemotherapy for Bcelllymphoma 4 detection of polymorphisms in human mitochondrial DNA 49. Which locus is used to identify the gender of the individual from whom a DNA sample May is obtained? a amelogenin b CSFIPO « TPOX a vWA 50. Which statement is true concerning molecular-based paternity testing? PNY a paternity testing is typically performed using VNTR loci in the mother, child and alleged father & single mismatch between an allele in the child (that must have come from the father of the child) and the alleged father is sufficient to exclude the alleged father ‘e match between the alleged father and the child ata rare allele carries less weight in paternity than does a match involving a higher frequency allele 4 paternity index is calculated for the alleged father using all loci tested, even if one of the loci does not directly match the child 376 The Board of Certification Study Galde5 a z vuavunuonenves Wem vvasevvenusaeus Svevsvussuaveooe HAM IMEeresonanenere E | |Answi SY ie ‘Mole oe 6: Molecular Biology | Molecular Science Answers Molecular Science 1b Spontaneous deamination of methyi- thiy cytosine results in thymine formation; it ‘occurs 2.5x more frequently than any other single nucleotide mutation. [Nuss 207, 178) 2d TheG2phase of the cell cycle follows the S phase, where DNA content goes from 2N to 4N or from diploid to tetraploid. ching 2007, p41 3a Heteronuclear RNA is made up of. newly transcribed mRNA that still contains intervening sequences. (uckinghson 200, p32) 4. €_ Methylation of CpG islands isan MASy_ epigenetic mechanism of gene silencing WBuhigh 207 p31] 5b Chromosomes with clearly defined short and long arms are known as submetacentric chromosomes [Backing 2007, 91591 6 € RT synthesizes a DNA molecule from May an RNA template {Noh 207 6) 7 € One X chromosome is passed on to all sons by the mother; since she is a carrier ofa mutant X allele, 50% of her sons will be affected. IWusstoum 2007, p32) 8d Males are hemiaygous for X-linked alleles because they have only one copy of the X chromosome, (ackingha 20,9130) 9d All mitochondria are passed on feom a mother to her children, but not by a father to his children IBckinghan 2007, p20} Molecular Techniques 10 ¢ ~The greater the concentration of 2, garose the smaller the sizeof DNA that can be resolved. Ueighm 207 92 878 The Board of Certification Study Guide it B a at B is 17 'b RFLP analysis is performed on DNA Southern blotting is the method used to separate DNA fragments by size, transfer the DNA fragments to a solid support, and then hybridize with a probe specific for the gene of interest. (Buckingham 2007, 9104) 4A reverse dot blot allows for the screening of several different mutations at one time by putting probes specific for different mutations on the membrane and then hybridizing a labeled patient DNA sample to the membrane Ucn 207, 79-80) High temperature and low salt concentration in the wash buffer favors dissociation of the probe: DNA targets that are not completely base-paired along the length of the probe. This will give the lowest nonspecific or background hybridization {cknghon 207, p07 1b Klenow fragment lacks 5'-»3" exonuclease activity and is therefore used for eandom primer labeling Wochingh 207, p10 b Agel mobility shift assay involves ‘mixing a labeled oligonucleotide with a protein extract to sev if specific protein: ‘nucleic acid interaction occurs. The oligonucleotide will exhibit an apparent increase in molecular size on a gel due to its association with a protein should such an interaction occur [ahaglam 2007, p17 118 b Alpha satellite probes are centromere specific and can be used ro identify specific chromosomes. A chromosome 21 specific alpha satelite probe would give 3 distinct dots in interphase cells, Chromosome paint probes have greater utility in metaphase spreads to detect translocations or other alterations in chromosome structure [A.nique sequence probe specific for chromosome 21 could also theoretically bbe used, but would give a weaker signal compared to the alpha satellite probe due to the highly repetitive nature of the alpha satellite sequence. {Buckingham 207, € volumes of ethanol are used to precipitate DNA, whereas 2.5 volumes are used to precipitate RNA. Buckingham 2007, 686: Molecular Biology | Applications of Molecular Testing 18 19 20 28 2, ‘€ _ Chelex® resin is often used to extract DNA from forensic samples, but may also be used for clinical samples. Bucinghar 207, 711 4 SYBR* Green is the most sensitive of the listed dyes. (Buckingham 2007, 7-70, € The absorptivity constant for DNA is 50 ng/mt. (Buckinghaes 2007, 977) b (OD260»40x40)/2 = (0.03125 » 40x 40)/2 =50/2 » 25 yg. Ibuckingham 2007, 977) € Strata particular locus differ in size, [Buckingham 2007, 9213) ¢ Reporter and quencher fluorochromes are separated from one another when the beacon anneals its target. [acing 207, p40) ‘4 _ Hydrogen bonds hold the 2 strands of DNA together ina double-stranded DNA molecule. {Mssdur 2007, 971 b Annealing temperature determines the amount of mismatch tolerated between the primers and the target IMuckinghsm 2007, p123) ‘PCR reactions in which multiple loci are amplified in the same reaction by multiple sets of primers are known as multiplex PCR, [Pekinghsm 2009, p34) € When target sequences are limited, 2 ‘successive PCR reactions can be performed using primer pairs that are 3'to the first set of primers in the second round of PCR. This is called a nested PCR reaction (uchingham 207, p36) FRET stands for fluorescent resonance energy transfer. One probe has a donor Auorophore at its 5’ end and the other has a reporter fluorophore a its 3’ end, When both probes anneal to one strand of an amplicon, the donor and reporter fuorophores achieve the correct geometry to generate a signal. [Buckingham 2007, 142] Answers 29 & Branched DNA analysis is performed iy. by capturing a target nucleic acid onto ‘microtiter plate and then adding an amplifier probe to greatly increase the signal resulting from the captured nucleic acid. PCR and NASB amplifications amplify the target nucleic acid sequence, whereas the ligase chain reaction amplifies the amount of probe in the reaction when the target is present. Backs 2007 p47) b The amplicon from X would not be digested by Alul because it lacke the enzyme recognition site and thus remains 176 bp in size. The amplicon for allele x would give the 118 bp and 58 bp fragments. Since the patient has bands of all 3 sizes on the gel he/she must be heterozygous, having one X allele arid one x allele, eka 207, p89) 32 € _ Bisulfite sequencing is a modified chain terminator method specifically designed to detect methylated nucleotides. IBuckingham 207,216) Fa Ps 2 d_SSCPisa screening test in which the Bhi, exact nature of a mutation does not need to bbe known in order to detect the presence of a mutation, {ackinghan 2007, p19) 33 b UNG digests amplicons containing itiiy dUTP as part of a system to prevent contamination of PCR products generated in previous rounds of PCR in a lab, cing 2007, 913] 34. & When primers span an in exon-intron- [ihiv exon boundary, amplicons generated from genomic DNA would be larger than the specific amplicon generated from a mature mRNA, Icleman 2006 p10 Applications of Molecular Testing 35. € The HIV genome is RNA, so only NABSA ONly would allow direct detection. [Backing 2007, p26] 36 € PPGE after digestion of bacterial Suv. chromosomes with restriction eneymes is used to determine similarity between bacterial ieolates (Bunga 200, 28-26) (Clinical Laboratory Certification Bxaminations 379 |6: Molecular Biology | Applications of Molecular Testing Answers ‘Bacterial genotyping by PEGE follows the rule of three, s0 3 or more differences indicate the test strain is different from the reference strain. {Buckingham 2007, 9286] 4 Human DNA (extrinsic to the target DNA) would be expected to be collected along with the Mycoplasma DNA (intrinsic to the sample). [Buckingham 207, p267] ‘2 15481 is found in both B pertussis and B homesii and 151001 is found in B homesit and B parapertussis, but not in 8 pertussis. 18481 is not found in B parapertussis. {Buckingham 2007, 92791 € mech is the gene responsible for resistance in Staph aureus; vanA for vancomycin resistance in Enterococcus; inhA for isoniazid in Mycobacterium tuberculosis; and rpeB for rifampin in Mycobacterium tuberculosis. {Buckingham 2007p 282) € The 922 translocation creates the BCR/ABL1 fusion gene in CML, ‘oak 2007, A181 b Only 16S ribosomal RNA is found in prokaryotes and is the target; the others. listed are eukaryotic RNA subunits. {Buckingham 2007, 9272) The presence of a distinct band along ‘with the smear indicates atleast 1% of, the patient's Bcells have a monoclonal rearrangement of the lg heavy chain. Iuckiogham 2007, 93521, bh Turner syndrome females have a 45X genotype. (Wessun 2007, 9108) b Robertsonian translocations involving. chromosome 21q account for about 4% of the cases of Down syndrome. [Buckingham 2007, 9) 4 Sequence-specific PCR in 96-well plates is commonly performed to determine polymorphisms in class [and class II DRB and DRQ genes. (Buckingham 2007, 93901 Bb _AF2 could possibly have contributed all of the alleles inherited by the child after the alleles the child inherited from the mother have been considered. (Bucingbae 2007, 929) ‘Tae Board of Certification Study Galde 48d AVLand HV2 are portions of the {ky mitochondrial genome that are sequenced to determine mitochondrial inheritance, {Bucneghan 2007, 92581 49a Amelogenin gives different sized 1, amplicons based on whether itis amplified from the X or ¥ chromosomes, so males ‘would have 2 bands on a gel at the amelogenin locus, while females would have only 1. IBackingho 207, pp286288) 50 d_ A paternity index is calculated even Miiy with 1 oe 2 mismatches because germ line mutations could have occurred during the formation of sperm in the father. Ucn 2007, 2391