Molecular Diagnostics- Molecular methods

Question 1

1. Which double-stranded DNA molecule has the
   highest melting temperature?
   A. An oligonucleotide with a repeating sequence of
   A-A-A at the 5´ end
   B. A molecule of 5,000 base pairs with a high
   number of A-T base pairs
   C. An oligonucleotide with a large number of
   repeating C-G-C codons
   D. A DNA polymer of 100,000 base pairs

Answer 1

C. An oligonucleotide with a large number of
repeating C-G-C codons

Question 2

2. Which base pair sequence is most likely to serve as
   a binding site for a restriction endonuclease?
   A. A-T-T-C-A
   T-A-A-G-T
   B. C-T-A-C-T-G
   G-A-T-G-A-C
   C. C-A-C
   G-T-G
   D. A-A-G-C-T-T
   T-T-C-G-A-A

Answer 2

D. A-A-G-C-T-T
T-T-C-G-A-A

Question 3

3. Cloning a human gene into a bacterium in order
   to make a large molecular probe requires which
   vector?
   A. Plasmid
   B. Bacterial microsome
   C. 30S bacterial ribosome
   D. Single-stranded DNA

Answer 3

A. Plasmid

Question 4

4. What process can be used to make a DNA probe
   produce a fluorescent or chemiluminescent signal?
   A. Enzymatic attachment of acridinium esters to
   terminal ends of the probe
   B. Substitution of biotinylated or fluorescent
   nucleotides into the probe
   C. Splicing the gene for β-galactosidase into the
   probe
   D. Heat denaturation of the probe followed by acid
   treatment

Answer 4

B. Substitution of biotinylated or fluorescent
nucleotides into the probe

Question 5

5. What term describes the products produced when
   DNA is digested by restriction endonucleases?
   A. Mosaicisms
   B. Chimeras
   C. Amplicons
   D. Restriction fragment length polymorphisms

Answer 5

D. Restriction fragment length polymorphisms

Question 6

6. The following figure shows a DNA size standard
   (ladder) made by restriction enzyme digestion
   (PstI) of lambda phage DNA that has been
   separated by agarose gel electrophoresis. Which DNA band has the highest molecular weight ?

A. 1
B. 2
C. 3
D. 4

Answer 6

A. 1

Question 7

7. What reagent is most commonly used to stain
   DNA separated by electrophoresis?
   A. Silver nitrate
   B. Nicotinamide adenine dinucleotide
   C. Cationic dye
   D. Ethidium bromide

Answer 7

D. Ethidium bromide

Question 8

8. Which technique is used to detect DNA
   containing a specific base sequence by applying a
   labeled probe to DNA bands immobilized onto
   nitrocellulose paper following electrophoresis?
   A. Southern blot
   B. Northern blot
   C. Dot blot
   D. Western blot

Answer 8

A. Southern blot

Question 9

9. Which of the following types of mutation causes
   the premature termination of protein synthesis?
   A. Missense
   B. Nonsense
   C. Insertion
   D. Frame shift

Answer 9

B. Nonsense

Question 10

10. In humans, which component of a gene is
    translated into a protein?
    A. Intron
    B. Exon
    C. Promoter
    D. TATA box

Answer 10

B. Exon

Question 11

11. Which statement best describes a DNA
    polymorphism?
    A. A point mutation arising in a gene
    B. Any change in DNA that is associated with
    abnormal function
    C. A change in the base sequence of DNA that is
    translated into an abnormal protein
    D. A variation in DNA that occurs with a frequency
    of at least 1%

Answer 11

D. A variation in DNA that occurs with a frequency
of at least 1%

Question 12

12. Which of the following is the most common type
    of polymorphism?
    A. Single nucleotide polymorphism (SNP)
    B. Variable number tandem repeat (VNTR)
    C. Short tandem repeat (STR)
    D. Short repetitive interspersed element (SINES)

Answer 12

A. Single nucleotide polymorphism (SNP)

Question 13

13. Which of the following mechanisms facilitates
    DNA separation by capillary electrophoresis?
    A. Molecular sieving
    B. Partitioning
    C. Adsorption
    D. Deflection

Answer 13

A. Molecular sieving

Question 14

14. The polymerase chain reaction (PCR) involves
    three processes. Select the order in which these
    occur.
    A. Extension→Annealing→Denaturation
    B. Annealing→Denaturation→Extension
    C. Denaturation→Annealing→Extension
    D. Denaturation→Extension→Annealing

Answer 14

C. Denaturation→Annealing→Extension

Question 15

15. In the PCR cycle, how is denaturation
    accomplished?
    A. Heat
    B. Alkali treatment
    C. Addition of sulfonylurea
    D. Formamide

Answer 15

A. Heat

Question 16

16. What is the composition of the primer used
    in PCR?
    A. A cocktail of enzymes and nucleotide
    triphosphates that bind to the target
    B. An oligonucleotide complementary to bases at
    the 3´ end of the target
    C. A small piece of dsDNA that attaches to the
    template
    D. A probe made of mRNA that binds downstream
    from the target

Answer 16

B. An oligonucleotide complementary to bases at
the 3´ end of the target

Question 17

17. The master mix solution used for PCR contains
    which of the following reagents?
    A. Deoxyribonucleotide triphosphates
    B. Deoxyribonucleotide monophosphates
    C. Deoxyribonucleosides
    D. Ribonucleotide monophosphates

Answer 17

A. Deoxyribonucleotide triphosphates

Question 18

18. What is the unique characteristic of the DNA
    polymerase, Taq DNA polymerase, used in PCR?
    A. It can be enzyme labeled
    B. It is more efficient than eukaryotic polymerases
    C. It is heat stable
    D. It works with DNA of any species

Answer 18

C. It is heat stable

Question 19

19. In PCR methods, how can several targets be
    copied simultaneously and detected?
    A. By following the increase in absorbance at
    260 nm during melting
    B. By labeling multiple primers with specific fluors
    C. By substitution of hybridization probes for
    primers
    D. By analysis of adenosine tail signatures

Answer 19

B. By labeling multiple primers with specific fluors

Question 20

20. Which formula predicts the number of PCR
    products that can be produced?
    A. 2n where n is the number of cycles
    B. N4 where N is the number of cycles
    C. p2 + 2pq + q2 = 1 where p and q are the number
    of primers
    D. N2/2 where N is the number of cycles

Answer 20

A. 2n where n is the number of cycles

Question 21

21. How can PCR be applied to the detection of
    human immunodeficiency and other RNA viruses?
    A. The virus must be inserted into human DNA by
    viral integrase prior to PCR
    B. Substitute deoxyuridine triphosphate in place of
    deoxythymidine triphosphate in the master mix
    C. Add a heat-stable reverse transcriptase enzyme to
    the master mix
    D. Substitute ribonucleotide triphosphates for
    deoxyribonucleotide triphosphates in the
    master mix

Answer 21

C. Add a heat-stable reverse transcriptase enzyme to
the master mix

Question 22

22. Which statement best describes the method of
    branched DNA signal amplification?
    A. The DNA template is amplified directly using
    patented enzymes
    B. Multiple primers are used to create branches of
    the template DNA, permitting multiple
    extension sites
    C. The target DNA is denatured and hybridized to
    RNA, and the hybrid molecules are amplified by
    both DNA and RNA polymerases
    D. The target DNA is bound by multiple
    probes, and those are amplified instead of
    the target DNA

Answer 22

D. The target DNA is bound by multiple
probes, and those are amplified instead of
the target DNA

Question 23

23. A PCR reaction is performed, and the negative
    control demonstrates the presence of a detectable
    number of PCR products (amplicons) by capillary
    electrophoresis. What is the most likely cause?
    A. False-positive post-PCR hybridization reaction
    due to low stringency
    B. Dimerization of PCR primers
    C. Contamination of control sample with a trace
    amount of template DNA
    D. Background signal from gel fluorescence or
    inadequate removal of unbound probe

Answer 23

C. Contamination of control sample with a trace
amount of template DNA

Question 24

24. How can a false-negative PCR test caused by the
    presence of an inhibitor of the reaction in a
    patient’s sample be detected?
    A. Using a positive control
    B. Using an internal control
    C. Performing each test in duplicate
    D. Performing serial dilutions of the sample

Answer 24

B. Using an internal control

Question 25

25. All of the following are requirements for reducing contamination in DNA amplification methods except: A. Use of aerosol barrier pipette tips when transferring samples or reaction products B. Preparation of reagents in a dead air box or biological cabinet C. A separate area for performing preamplification, postamplification, and detection steps D. Pretreatment of samples with high-intensity ultraviolet light

Answer 25

D. Pretreatment of samples with high-intensity ultraviolet light

Question 26

26. Which method has been used successfully to reduce contamination in the preamplification stage of PCR? A. Substitution of deoxyuridine triphosphate for deoxythymidine triphosphate in the master mix B. Use of low-molecular-size primers C. Use of a denaturation temperature above 95°C D. Pretreatment of samples with antisense RNA

Answer 26

A. Substitution of deoxyuridine triphosphate for deoxythymidine triphosphate in the master mix

Question 27

27. How are PCR methods adapted to yield quantitative data? A. By comparing PCR product to an internal standard B. By applying a conversion factor to the PCR signal that converts it to copies per milliliter C. By determining the mass of PCR product using ultraviolet spectrophotometry D. By making serial dilutions of the sample

Answer 27

A. By comparing PCR product to an internal standard

Question 28

28. A PCR analysis of a vaginal sample for Chlamydia trachomatis gives a negative result (optical density of biotinylated reaction product below the cutoff point). The internal control result is also below the cutoff. Positive and negative controls produced acceptable results. What action should be taken? A. The test should be reported as negative B. The sample should be diluted and the test repeated C. The result should not be reported and the sample should be repeated D. A preliminary result of negative should be reported but should be confirmed by further testing using a different method of analysis

Answer 28

C. The result should not be reported and the sample should be repeated

Question 29

29. In real-time PCR analysis, the absolute concentration of PCR product is determined by plotting which two values? A. Fluorescent intensity versus melting temperature B. The threshold cycle versus concentration C. The well factor versus threshold cycle D. The melting temperature versus concentration

Answer 29

B. The threshold cycle versus concentration

Question 30

30. In real-time PCR, quantitation can be done without standards of known copy number. Relative quantitation (estimated concentration) is possible because: A. Each cycle generates a twofold increase in product B. Each cycle threshold represents a 10-fold increase in product C. The fluorescence of two samples can be compared directly D. Concentration is proportional to fluorescence at the endpoint of the PCR reaction

Answer 30

A. Each cycle generates a twofold increase in product

Question 31

31. Which real-time PCR parameter can be used to detect the presence of a contaminant? A. Threshold cycle B. Baseline C. Melting temperature D. Relative fluorescent intensity

Answer 31

C. Melting temperature

Question 32

32. In real-time PCR, what value is needed in order to determine the threshold? A. Background signal B. Melting temperature C. Maximum fluorescence D. Threshold cycle

Answer 32

A. Background signal

Question 33

33. In real-time PCR, which of the following methods is not based on using a probe? A. TaqMan B. Molecular beacon C. Scorpion D. SYBR green

Answer 33

D. SYBR green

Question 34

34. Which statement accurately describes the process of fluorescent in situ hybridization (FISH)? A. Hybridization is performed on DNA extracted from cells B. Hybridization is performed directly on intact chromosomes C. Hybridization probes are attached to histones associated with the chromosomes D. Hybridization occurs by attachment to the probe only at the centromere

Answer 34

B. Hybridization is performed directly on intact chromosomes

Question 35

35. Which type of specimen would be unsuitable for FISH analysis? A. Paraffin-embedded tissue B. Cells with chromosomes in metaphase C. Cells with chromosomes in interphase D. A cell suspension containing maternal and fetal blood

Answer 35

D. A cell suspension containing maternal and fetal blood

Question 36

36. FISH can distinguish each of the following chromosomal abnormalities except: A. Aneuploidy B. Translocation C. Deletion D. Trinucleotide repeats

Answer 36

D. Trinucleotide repeats

Question 37

37. In microarray and macroarray analysis, which molecules are labeled? A. The immobilized DNA molecules B. The sample DNA C. Both target and sample molecules D. The substrate matrix

Answer 37

B. The sample DNA

Question 38

38. How can all of the mRNA within a sample be amplified to prepare microarray probes? A. A specific primer for each mRNA must be synthesized B. A primer is made to the polyA tail of mRNA C. Nonspecific attachment of T7 polymerase occurs when the cells are treated with detergent D. Random primer sets are used under low stringency conditions

Answer 38

B. A primer is made to the polyA tail of mRNA

Question 39

39. What is the difference between a microarray and a macroarray DNA assay? A. The number of targets is larger on a macroarray B. The molecular size of each target is larger on a macroarray C. The amount of each target is larger on a macroarray D. The substrate used for a macroarray is different from a microarray

Answer 39

C. The amount of each target is larger on a macroarray

Question 40

40. Protein microarray analysis requires the use of which of the following techniques to generate protein profile data? A. Electrophoresis B. Mass spectroscopy C. Thin-layer chromatography D. Gas chromatography

Answer 40

B. Mass spectroscopy