Module 4 FISH Flashcards
Which of the following counterstain dyes should be used to counterstain the cells with
Spectrum Green /Spectrum Red dual color signals?
A. DAPI
B. Propidium iodine C. Ethidium bromide D. Acridine orange
A
DAPI is a _________ counterstain.
A. green B. yellow C. blue D. red
C
The process of fluorescence in situ hybridization is used to:
A. Structure a complete karyotype. B. Show the repair of DNA sequences after denaturation. C. Visualize specific sequence of DNA with a fluorochrome. D. Label colonies of cultured cells.
C
Which of the following is critical to the success of a FISH procedure?
A. Probe can only be labeled with the fluorochrome. B. Temperature of the denature solution. C. Slides should be aged at 60oC for at least overnight. D. The FISH signal should be detected immediately after the hybridization.
B
Formamide is used in the FISH procedure for which of the following purposes:
A. To be used to rinse off excess salt from the slides. B. To modify a DNA probe so it can be detected by a fluorochrome. C. To artificially age the slide. D. To lower the DNA melting point.
D
The purpose of the post hybridization wash is to:
A. Enhance the binding of the fluorochrome B. Serve as a pretreatment for probe labeling C. Remove unbound probe from the slide D. All of the above
C
Which of the following is true about slide preparation for FISH?
A. Slides should be kept at room conditions.
B. Slides should be treated overnight in a 90oC oven. C. Slides should be prepared 1 week in advance and stored in the refrigerator. D. Slides should be protected from ultraviolet light
A
When analyzing a sample stained with a FISH probe, the technologist notices a high amount of nonspecific cross-hybridization. Which of the following is the most likely cause?
A. The counterstain was not added. B. The wash stringency was too low. C. The manufacturer produced a poor probe set. D. The denaturation process did not work.
B
When using the Applied Imaging system to capture the FISH image, which program should be chosen?
A. Fluorescent
B. Probe C. RXFISH D. MFISH
B
Which of the following statements is true about directly labeled probes?
A. Should be counterstained only with DAPI.
B. Require amplification of the probe signal. C. Exposure to light should be avoided. D. Denaturation of the probe is not necessary.
C
Which of the following is critical to the success of a FISH procedure?
A. Probe can only be labeled with the biotin hapten B. Temperature of the denaturation solution C. Slides should be aged at 60oC for at least one week D. The FISH signal should be detected immediately after the hybridization procedure.
B
The hapten digoxigenin is used in the FISH procedure for which of the following purposes?
A. To provide a binding site for fluorochromes, thereby allowing probe detection.
B. To artificially age the slide C. To provide a fluorescent signal that can be seen with a fluorescent microscope D. To be used to rinse off excess salt and formamide from the slide.
A
Formamide is used in the FISH for which of the following purposes?
A. To be used to rinse off excess salt from the slide B. To modify a DNA probe so it can be detected by a fluorochrome C. To artificially age the slide D. To denature the DNA prior to hybridization
D
The intensity of probe signal and the amount of background is dependent on the stringency level of which of the following chemicals?
A. SSC
B. Digoxigenin C. Phosphate buffer D. Propidium iodide
A
The M-FISH procedure makes use of combinatorial labeling in order to uniquely label all chromosomes human chromosomes. Which of the following represents the correct mathematical equation for determining the number of combinations based on the number of fluorochromes (n) available?
A. # of Combinations = (2n-1) B. # of Combinations = (n-1/2)(2) C. # of Combinations = n-12 D. # of Combinations = (2n)
A
What is the maximum number of color combinations that can be obtained from 5 fluorochromes?
A. 7 B. 15 C. 31 D. 63
C
DAPI is a:
A. red counterstain B. high stringency wash C. low stringency wash D. blue counterstain
D
Propidium iodide is a:
A. blue counterstain B. red counterstain C. high stringency wash D. low stringency wash
B
The purpose of the post-hybridization wash is to:
A. Enhance the binding of the fluorochrome B. Serve as a pretreatment for probe labeling C. Remove unbound probe from the slide D. Enhance RNase removal from the slide
C
Why are whole chromosome probes and FISH not suitable at this time for detection of mosaic trisomies in interphase nuclei?
A. The probes cannot penetrate through the interphase nuclei to the DNA. B. The domains can overlap each other in a two dimensional field. C. More than two signals appear routinely in more than 50% of normal cells.
B
Interphase cytogenetic analysis using chromosome-specific probes is most useful for:
A. Detecting mosaicism B. Detecting chromosomal rearrangements C. Providing a rapid diagnosis for a suspected numerical chromosome abnormality. D. Detecting single gene disorders
C
Which of the following IS NOT an advantage of FISH diagnostics concerning interphase testing?
A. Is more informative than chromosome karyotyping
B. Can rapidly detect sex chromosomes or aneuploidy of chromosomes. C. Requires no culture. D. Requires only simple microscope analysis.
A
In situ hybridization was originally introduced as a method which detects:
A. The abnormal rearrangement of genes B. Highly repetitive nucleic acid sequences C. Highly repetitive genes D. Highly unusual nucleic acid sequences
B
The technologist responsible for the FISH procedure has repeated problems with poor chromosome spreading due to cytoplasm remaining around the chromosomes. How will this cytoplasm affect the FISH procedure?
A. Hybridization of the probe to its target DNA is unaffected by cytoplasm. B. Detection of the probe with appropriate fluorochrome is unaffected by cytoplasm. C. When detected with the appropriate fluorochrome there will be a decrease in background signal. D. There will be a significant reduction or total lack of hybridization of the probe to its target DNA.
D
50% post wash at 37oC:
A. is necessary for probe denaturation B. is a high stringency wash C. is necessary for DNA denaturation D. is a low stringency wash
D
50% post wash at 72oC:
A. is necessary for fluorescent detection B. is necessary for counterstaining C. is a high stringency wash D. is a low stringency wash
C
Which one(s) might contribute to poor hybridization or a weak signal?
A. denaturing probe at the wrong temperature B. cytoplasm C. denaturing slide at the wrong temperature D. all of the above
D
The temperature of 72oC is necessary in which of the following FISH procedures?
A. simultaneous denaturation B. probe storage C. standard denaturation D. PBD washing
C
The temperature of 37oC is necessary in which of the following FISH procedures?
A. PBD washing B. Formamide washing C. 2X SSC aging D. FITC-avidin storage
C
The temperature of 45oC is necessary in which of the following FISH procedures?
A. PBD washing
B. Formamide washing C. 2X SSC washing D. FITC-avidin storage
B
The temperature of 45oC is necessary in which of the following FISH procedures?
A. PBD washing
B. Formamide washing C. 2X SSC washing D. FITC-avidin storage
B
The temperature of -20oC is necessary in which of the following FISH procedures?
A. Probe storage
B. PBD washing C. simultaneous denaturation D. FITC-avidin storage
A
The temperature of 4oC is necessary in which of the following FISH procedures?
A. probe storage B. FITC-avidin storage C. PBD washing D. 2X SSC washing
B
The temperature of 90oC is necessary in which of the following FISH procedures?
A. standard denaturation B. formamide washing C. simultaneous denaturation D. 2X SSC washing
C
Room temperature is necessary in which of the following FISH procedures?
A. PBD washing
B. formamide washing C. 2X SSC washing D. FITC- avidin storage
A
Which of the following is NOT critical to the success of a FISH procedure?
A. Accessibility of the probe to the target B. Temperature of the denaturation solution C. Cells which have been dropped flat D. Cells aged at room temperature for at least three weeks
D
COT-1:
A. Serves as a pretreatment to increase accessibility of the probe. B. Removes repetitive sequences from the reaction mixture. C. Enhances binding of the fluorochrome to the reporter molecule. D. Conterstains the DNA after fluorescence detection.
B
Which of the following does NOT affect stringency?
A. Temperature B. Salt concentration C. Formamide concentration D. The type of probe used E. All affect stringency
E
Directly labeled probes:
A. Are counterstained immediately following the post hybridization wash
B. Utilize biotin as the reporter molecule C. Bind the fluorochrome after the post hybridization wash D. Require amplification of the probe signal
A
The observance of monosomy in an interphase cell may be due to:
A. Overlay of one probe hybridization spot onto another B. Incomplete penetration of the probe into the nucleus C. Incomplete denaturation or loss of target DNA D. all of the above
D
Slide sealing with rubber cement during the FISH procedure is usually done to:
A. Prevent drying (especially in a non-humidified incubator) B. Prevent absorption of water from the environment (especially in a chamber saturated with moisture) C. Both a and b D. Neither a or b
C
When performing the FISH procedure while testing for microdeletion syndromes it is necessary to have a positive control. Which of the following is TRUE concerning this assay?
A. For a positive control, one probe should be within the critical region and one probe should be outside the critical region on the same chromosome.
B. For a positive control, one probe should be within the critical region and one probe should be tested on another chromosome. C. For a positive control, normal individuals should be tested to determine that they do not show a deletion. D. For a positive control, the critical region probe should be tested and shown to be deleted in the majority of patients with the syndrome.
A
A report on a Prader Willi Syndrome case shows a cytogenetic deletion by conventional cytogenetics that is not confirmed with FISH testing. What is the likely reason for this discrepancy?
A. The probe is outdated B. The deletion is outside the critical region C. The FISH test is rarely used in determining microdeletion syndromes because of its low sensitivity. D. Both b and c E. all of the above
B
When using a fluorescein labeled probe during the FISH procedure, the appropriate counterstain used should be:
A. 5-Bromodeoxyuridine B. ethidium bromide C. propidium iodide D. DAPI
C
Some possible quality control measures that could be used for FISH studies are:
A. percentage of cells rejected per specimen B. results of the positive and/or negative control C. inter-observer variation on the same specimen D. both b and c E. all of the above
E
FISH with enumeration probes to screen for common aneuploidies should be used for:
A. all specimens submitted to the cytogenetic lab from spontaneous abortions B. all specimens submitted to the cytogenetic lab from molar pregnancies C. only specimens suspicious for sex abnormalities D. only specimens that fail to grow in culture or where maternal contamination is suspected
A
