PRELIMS Flashcards
Indicate whether the following peptides are hydrophilic
or hydrophobic:
a. MLWILSS
b. VAIKVLIL
c. CSKEGCPN
d. SSIQKNET
e. YAQKFQGRT
f. AAPLIWWA
g. SLKSSTGGQ
a. MLWILAA — hydrophobic
b. VAIKVLIL — hydrophobic
c. CSKEGCPN — hydrophilic
d. SSIQKNET — hydrophilic
e. YAQKFQGRT — hydrophilic
f. AAPLIWWA — hydrophobic
g. SLKSSTGGQ — hydrophilic
. Is the following peptide positively or negatively
charged at neutral pH?
GWWMNKCHAGHLNGVYYQGGTY
Answer: The peptide is positively charged
Consider an RNA template made from a 2:1 mixture
of C:A. What would be the three amino acids most
frequently incorporated into protein?
Answer: Proline (CCA), histidine (CAC), and
threonine (ACC) would be most frequently
incorporated.
What is the peptide sequence encoded in
AUAUAUAUAUAUAUA . . .?
Answer: The peptide sequence would alternate
isoleucine (AUA) and tyrosine (UAU):
IYIYI. . .
Write the anticodons 5’ to 3’ of the following amino
acids:
a. L
b. T
c. M
d. H
e. R
f. I
Answers:
a. L — UAA, CAA,AAG, GAG, UAG, CAG
b. T — AGU, GGU, UGU, CGU
c. M — CAU
d. H — AUG, GUG
e. R — ACG, GCG, UCG, CCG
f. I — AAU, GAU
A protein contains the sequence LGEKKW
CLRVNPKGLDESKDYLSLKSKYLLL. What is the likely function of this protein? (Note: See
Box A3-4.)
Answer: This protein has a leucine residue at se
quential 7th positions forming a leucine zipper,
found in transcription factors.
A histone-like protein contains the sequence:
PKKGSKKAVTKVQKKDGKKRKRSRK. What
characteristic of this sequence makes it likely to
associate with DNA?
This protein is positively charged, which would facilitate association with negatively charged DNA
A procedure for digestion of DNA with a restriction
enzyme includes a final incubation step of 5 minutes
at 95ºC. What is the likely purpose of this final step?
The 95ºC incubation will inactivate the protein, preventing its activity in subsequent
steps of the assay.
What is a ribozyme?
A ribozyme is an RNA molecule that can metabolize other molecules like an enzyme.
Name the nonprotein prosthetic groups for the
following conjugated proteins:
glycoprotein
lipoprotein
metalloprotein
glycoprotein — sugars
lipoprotein — lipids
metalloprotein — metal atoms
Calculate the DNA concentration in µg/mL from the following information:
a. Absorbance reading at 260 nm from a 1:100
dilution = 0.307
b. Absorbance reading at 260 nm from a 1:50
dilution = 0.307
c. Absorbance reading at 260 nm from a 1:100
dilution = 0.172
d. Absorbance reading at 260 nm from a 1:100
dilution = 0.088
a. 0.307 X 50 ug/mL = 15.35 ug/mL
15.35 ug/mL X 100 = 1535 ug/mL
b. 0.307 X 50 ug/mL = 15.35 ug/mL
15.35 ug/mL X 50 = 767.5 ug/mL
c. 0.172 X 50 ug/mL = 8.60 ug/mL
8.60 ug/mL X 100 = 860 ug/mL
d. 0.088 X 50 ug/mL = 4.40 ug/mL
4.40 ug/mL X 100 = 440 ug/mL
If the volume of the above DNA solutions was
0.5 mL, calculate the yield for (a) – (d)
a. 1535 ug/mL 0.5 mL = 767.5ug
b. 767.5 ug/mL 0.5 mL = 383.8 ug
c. 860 ug/mL 0.5 mL = 430 ug
d. 440 ug/mL 0.5 mL = 220 u
After agarose gel electrophoresis, a 0.5 µg aliquot of DNA isolated from a bacterial culture produced only a faint smear at the bottom of the gel lane. Is this an acceptable DNA sample?
This amount of bacterial DNA should produce a bright smear near the top of the gel lane. This DNA is probably degraded and is therefore unacceptable.
Contrast the measurement of DNA concentration by
spectrophotometry with analysis by fluorometry with
regard to staining requirements and accuracy.
Spectrophotometry requires no DNA staining. Fluorometry requires staining of DNA to
generate a fluorescent signal. Fluorometry may
be more accurate than spectrophotometry, since
double-stranded DNA must be intact to stain and
generate a signal, whereas single nucleotides will
absorb light in spectrophotometry.
Calculate the RNA concentration in µg/mL from the
following information:
a. Absorbance reading at 260 nm from a 1:100
dilution = 0.307.
b. Absorbance reading at 260 nm from a 1:50
dilution = 0.307.
c. Absorbance reading at 260 nm from a 1:100
dilution = 0.172.
d. Absorbance reading at 260 nm from a 1:100
dilution = 0.088.
a. 0.307 X 40 ug/mL = 12.28 ug/mL
12.28 ug/mL X 100 = 1228 ug/mL
b. 0.307 X 40 ug/mL = 12.28 ug/mL
12.28 ug/mL X 50 = 614 ug/mL
c. 0.172 X 40 ug/mL = 6.88 ug/mL
6.88 ug/mL X 100 = 688 ug/mL
d. 0.088 X 40 ug/mL = 3.52 ug/mL
3.52 ug/mL X 100 = 352 ug/mL
An RNA preparation has the following absorbance
readings:
A260 = 0.208
A280 = 0.096
Is this RNA preparation satisfactory for use?
The A260/A280 ratio is 0.208/0.096 = 2.17.
This RNA preparation is satisfactory for use.
A blood sample was held at room temperature for
5 days before being processed for RNA isolation. Will this sample likely yield optimal RNA?
This sample will likely not yield optimal RNA due to degradation and changes in gene expression at room temperature.
Name three factors that will affect yield of RNA from a paraffin-embedded tissue sample.
Isolation of RNA from fixed tissue is especially affected by the type of fixative used, the age/length of storage of the tissue, and the preliminary handling of the original specimen
You wish to perform an electrophoretic resolution of
your restriction enzyme–digested DNA. The size of
the expected fragments ranges from 100 to 500 bp.
You discover two agarose gels polymerizing on the
bench. One is 0.5% agarose; the other is 2% agarose.
Which one might you use to resolve your fragments?
The 2% agarose is best for this range of
fragment sizes.
After completion of the electrophoresis of DNA frag
ments along with the proper molecular-weight stan
dard on an agarose gel, suppose (a) or (b) below was
observed. What might be explanations for these?
a. The gel is blank (no bands, no molecular-weight
standard).
b. Only the molecular weight standard is visible.
a. Since the molecular-weight standard is not
visible, something is wrong with the general
electrophoresis process. Most likely, staining
with ethidium bromide was omitted.
b. The presence of the molecular-weight standard
indicates that the electrophoresis and staining
were performed properly. In this case, the
DNA fragments were not loaded, or the
method used to produce the fragments was
unsuccessful
How does PFGE separate larger fragments more efficiently than standard electrophoresis?
PFGE forces large fragments through
the gel matix by repeatedly changing the direction
of the electric current, thus realigning the sample
with spaces in the gel matrix.
A 6% solution of 19:1 acylamide is mixed, deaerated,
and poured between glass plates for gel formation.
After an hour, the solution is still liquid. What might
be one explanation for the gel not polymerizing?
The nucleating agent and/or the polymerization catalyst were not added to the gel solution.
A gel separation of RNA yields aberrantly migrating
bands and smears. Suggest two possible explanations
for this observation
This RNA could be degraded. Alternatively, improper gel conditions were used to separate the RNA.
Why does DNA not resolve well in solution (without
a gel matrix)?
Particles move in solution based on their
charge/mass ratio. As the mass of DNA increases,
slowing migration, its negative charge increases,
counteracting the effect of mass.
Why is SyBr green I less toxic than EtBr
SyBr green is a minor groove binding
dye. It does not disrupt the nucleotide sequence of
DNA. EtBr is an intercalating agent that slides in
between the nucleotide bases in the DNA and can
cause changes in the nucleotide sequence (mutations) in DNA.
What are the general components of loading buffer
used for introducing DNA samples to submarine gels?
Gel loading buffer contains a density
agent to facilitate loading of sample into the wells
underneath the buffer surface and a tracking
dye to follow the migration of the DNA during
electrophoresis.
Name two dyes that are used to monitor migration of
nucleic acid during electrophoresis.
Bromphenol blue and xylene cyanol
green are two of several tracking dyes.
When a DNA fragment is resolved by slab gel electrophoresis, a single sharp band is obtained. What is the
equivalent observation had this fragment been fluorescently labeled and resolved by capillary electrophoresis?
Capillary electrophoresis results will be
a single peak on an electropherogram.
A master mix of all components (except template)
necessary for PCR contains what basic ingredients?
Components of a standard PCR reaction mix include dNTPs, oligonucleotide primers, a
buffer of proper pH and mono- and divalent cation concentration, and polymerase enzyme.
The final concentration of Taqpolymerase is to be
0.01 units/µL in a 50 µL PCR. If the enzyme is
supplied as 5 units/µL,how much enzyme would you
add to the reaction?
a. 1 µL
b. 1 µL of a 1:10 dilution of Taq
c. 5 µL of a 1:10 dilution of Taq
d. 2 µL
Use VCVC to determine the volume of
enzyme:
0.01 50 = 5 x
x= (0.01 50)/5 = 0.1 µL
Option b would deliver 0.1 µL enzyme.
Primer dimers result from
Primer dimmers occur when primers hybridize to each other at the 3’ ends (option d).
Which control is run to detect contamination in PCR?
A reaction mix with all components except template (reagent blank, d) is used to
monitor for contamination.
Answer: Positive, negative, Molecular weight marker
Nonspecific extra PCR products can result from
a. mispriming
b. high annealing temperatures
c. high agarose gel concentrations
d. omission of MgCl2 from the PCR
Nonspecific products occur when
primers bind to regions other than the intended
target (a). Omission of magnesium could possibly
produce mispriming but more likely will prevent enzyme function.
Using which of the following is an appropriate way to
avoid PCR contamination?
a. High-fidelity polymerase
b. Hot-start PCR
c. A separate area for PCR setup
d. Fewer PCR cycles
A separate area for PCR setup
What are the three steps of a standard PCR cycle?
The three steps of the PCR cycle are denaturation, annealing, and extension.
How many copies of a target are made after 30 cycle of PCR?
2^30
Which of the following is a method for purifying a
PCR product?
a. Treat with uracil N glycosylase.
b. Add divalent cations.
c. Put the reaction mix through a spin column.
d. Add DEPC.
Spin columns (c) are used to purify PCR products from other components of the reaction mix.
In contrast to standard PCR, real-time PCR is
a. quantitative
b. qualitative
c. labor-intensive
d. sensitive
Quantitative
In real-time PCR, fluorescence is not generated by
which of the following?
a. FRET probes
b. TaqMan probes
c. SYBR green
d. Tth polymerase
Tth polymerase (d) does not fluoresce.
How does nested PCR differ from multiplex PCR?
Nested PCR requires two rounds of replication. Multiplex PCR is a single PCR reaction where more than one product is made using
multiple primer sets
What replaces heat denaturation in strand displacement amplification?
In strand displacement amplification, a nick in the double-stranded product is used to prime replication, rather than denaturation and primer binding.
An unlabeled collection tube with a requisition for a factor V Leiden test is received in the laboratory.
Notify the supervisor and reject the specimen.
After PCR, the amplification control has failed to yield a product.
Check the original DNA or RNA preparation. If it is adequate, repeat the amplification.
If not, reisolate the nucleic acid.
An isolated DNA sample is to be stored for at least
6 months.
Store at –70°C in a tightly sealed tube.
A bone marrow specimen arrives at the end of a shift
and will not be processed for the Bcl-2 translocation until the next day.
Place the specimen in the refrigerator.
The temperature of a refrigerator set at 8°C (±2°C)
reads 14°C.
Recheck the temperature after a few hours. If it does not return to range, notify the supervisor.
A PCR test for the BCR/ABL translocation was negative for the patient sample and for the sensitivity control.
Repeat the PCR with the addition of a new sensitivity control.
A fragile X test result has been properly reviewed and reported
File the test results, documents, and associated autoradiographs together in the laboratory archives.
A bottle of reagent alcohol with a 3 in the red diamond on its label is to be stored.
Place the alcohol bottle in a safety
storage cabinet for flammable liquids.
The expiration date on a reagent has passed.
Discard the reagent. If it can be used for nonclinical purposes, label and store it in a separate area away from patient testing reagents.
Test results are to be faxed to the ordering physician
Fax the results with a cover sheet containing the proper disclaimer.
Among a group of properly labeled containers, there
is a container similar to the others, but unlabeled.
Do not use the material in the unlabeled container. Consult the institutional safety representative for proper disposal of the contents.
A blood sample is received in a serum tube for PML-RARa testing by PCR
Notify the supervisor that the specimen cannot be tested and explain the collection error
(wrong/no anticoagulant).
The laboratory is reporting a gene mutation replacing glycine at position 215 with alanine. The results are written 215G→A in the test report.
Correct the test report to A215G before verification by the laboratory director.
The laser on a complex instrument malfunctions
Call the instrument manufacturer. Do not try to access the laser.
A test result falls outside of the analytical measurement range of the validated method
If the result exceeds the range, dilute the specimen and retest. If the results are below
the range, report the results as less than the lower limit of the assay.
Reference standards are reading higher than their designated concentrations.
Calibrate the instrument/detection system using the reference standards.
A new FDA-approved test does not perform as expected
Do not use the test. Contact the manufacturer. Consider an alternate test system
A solution containing radioactive waste has been
stored in a properly protected area for 10 half-lives
of the isotope.
The solution may be safely discarded;
however, contact the institutional safety department before discarding. Document all radioactive waste disposal.
There is no available proficiency test for a new method developed in the laboratory.
Establish an agreement with another laboratory to trade samples for testing. Use
split samples from reference standards. Compare results with a reference in-house method,
regional pools, previously assayed material, clinical evaluation, or other documented means
A research investigator asks for a patient specimen
Notify the supervisor. Do not release patient material without proper consent and
patient safety review board approval.
What characteristic of the genetic code facilitates identification of open reading frames in DNA sequences?
Out-of-frame or chance coding sequences tend to be short, often ending in a stop codon
Compare and contrast EIA with western blots for detection of protein targets
EIA is a liquid handling method and more easily automated than western blot. EIA is performed with immobilized antibodies exposed to test fluid. In western blot, antibodies or antigens are resolved by electrophoresis before exposure to test fluid.
On a size exclusion column, large molecules
will elute _______________ (before/after) small
molecules.
before
MALDI methods separate ions by
a. molecular volume
b. mass
c. charge
d. mass and charge
Mass and Charge
What is a heteroduplex?
A heteroduplex is a double-stranded nucleic acid with one or more noncomplementary
bases.
Which of the following methods would be practical to
use to screen a large gene for mutations?
a. SSP-PCR
b. SSCP
c. PCR-RFLP
d. DGGE
e. FP-TDI
DGGE (d) would be practical to use to screen a large gene for mutations as it can scan several hundred base pairs at a time.
SSCP is limited by the size and GC content of fragments that can be accurately analyzed. The remaining
methods are designed to detect known point
mutations
What is the effect on the protein when a codon sequence is changed from TCT to TCC?
There would be no effect on the protein as both TCT and TCC code for serine.
After an automated dye primer sequencing run, the
electropherogram displays consecutive peaks of the
following colors:
red, red, black, green, green, blue, black, red, green,
black, blue, blue, blue
TTGAACGTAGCCC
A dideoxy sequencing electropherogram displays
bright (high, wide) peaks of fluorescence, obliterating
some of the sequencing peaks. What is the most likely
cause of this observation? How might it be corrected
The likely cause is the presence of unincorporated labeled dideoxynucleotides or dye
blobs. Cleaning the sequencing ladder with spin
columns, ethanol precipitation, or bead binding
will correct this problem
In a manual sequencing reaction, the sequencing ladder on the polyacrylamide gel is very bright and read
able at the bottom of the gel, but the larger (slower migrating) fragments higher up are very faint. What is
the most likely cause of this observation? How might
it be corrected?
The loss of longer products is caused by an overly high dideoxynucleotide/deoxynucleotide
ratio. The problem can be corrected by lowering
the concentration of dideoxynucleotides in the
sequencing reaction.
Which of the following is not next-generation
sequencing?
a. 454 sequencing
b. tiled microarray
c. SOLID system
d. Illumina Genome Analyzer
b. tiled microarray
Which of the following projects would require mass array sequencing?
a. Mapping the hemochromatosis gene
b. Sequencing a viral genome
c. Characterization of a diverse microbial
population
d. Typing a single bacterial colony
Characterization of a diverse microbial population (c) would require high throughput
sequencing
Which of the following genes would be analyzed
to determine whether an isolate of Staphylococcus
aureus is resistant to oxacillin?
a. mecA
b. gyrA
c. inhA
d. vanA
mecA. S. aureus developed resistance to antibiotics that target its penicillin-binding
protein (PBP1) by replacing PBP1 with PBP2a encoded by the mecA gene. PBP2a found in
methicillin-resistant S. aureus (MRSA) has a low binding affinity for methicillin.
Which of the following is a genotypic method used
to compare two isolates in an epidemiological
investigation?
a. Biotyping
b. Serotyping
c. Ribotyping
d. Bacteriophage typing
Ribotyping
For which of the following organisms must caution be
exercised when evaluating positive PCR results
because the organism can be found as normal flora in
some patient populations?
a. Neisseria gonorrhoeae
b. HIV
c. Chlamydophila pneumoniae
d. Streptococcus pneumoniae
Streptococcus pneumoniae. Although PCR is specific for S. pneumoniae, the clinical significance of a positive PCR assay is question
able because a significant portion of the population (especially children) is colonized with the
organism and PCR cannot discern between colonization and infection.
Which of the following controls are critical for ensuring that amplification is occurring in a patient sample and that the lack of PCR product is not due to the
presence of inhibitors?
Amplification control. The amplification control should always generate a product, even in the absence of the target.
A PCR assay was performed to detect Bordetella per
tussis on sputum obtained from a 14-year-old girl who has had a chronic cough. The results revealed two bands, one consistent with the internal control and the other consistent with the size expected for amplification of the B. pertussis target. How should these
results be interpreted?
The girl has clinically significant B.
pertussis infection. Molecular-based assays are used almost exclusively for analysis of microorganisms such as N. gonorrhoeae, C. trachomatis, and B. pertussis.
Which of the following is a disadvantage of molecular
based testing?
a. Results stay positive longer after treatment than
do cultures.
b. Results are available within hours.
c. Only viable cells yield positive results.
d. Several milliliters of specimen must be submitted
for analysis.
Results stay positive longer after
treatment than do cultures. Molecular-based results can detect both living and dead organ
isms. A positive result due to dead or dying organisms complicates interpretation of
treatment efficacy.
A molecular-based typing method that has high typing capacity, reproducibility and discriminatory
power, moderate ease of performance, and good
to-moderate ease of interpretation is
a. repetitive elements
b. PFGE
c. plasmid analysis
d. PCR-RFLP
PFGE
A patient has antibodies against HCV and a viral
load of 100,000 copies/mL. What is the next test that
should be performed on this patient’s isolate?
a. Ribotyping
b. PCR-RFLP
c. Hybrid capture
d. Inno-LiPA HCV genotyping
Inno-LiPA HCV genotyping. The
viral load and the HCV genotype are used to determine the therapeutic protocol, both type
of drug(s) as well as duration. Methods available in the laboratory for HCV genotyping are PCR with RFLP analysis and reverse hybridization
A positive result for HPV type 16 indicates
a. high risk for antibiotic resistance
b. low risk for cervical cancer
c. high risk for cervical cancer
High risk for cervical cancer. Of
the sexually transmitted types of HPV, 12 to 15 oncogenic genotypes have strong association
with cervical cancers and are considered high
risk (HR) HPV types.
This method is recommended for smear preparations of bronchial aspirates and fresh sputum
A.Pull apart - serous fluid specimen
B. Smear preparation - blood smear
C. Touch preparation - aka impression technique
D. Spreading
Spreading
Which of the ff concentration is ideal and preferred for
glutaraldehyde in immunoelectron microscopy?
A. 0.10%
B. 3% - associated conc
C. 10%
D. 0.25%
0.25%
Fixation time can be cut down by using the ff, except:
A. Heat
B. Cold temp
C. Agitation
D. Microwave
Cold Temp
The majority of non-fatty unfixed tissues are
sectioned well at temperatures between ___ and ____.
-10C, -25C
Best fixative tissue for tissues containing iron
pigments?
A. Zenker’s Fluid - ginagamit more on the content of
mercury fluoride: liver, spleen, connective tissue
and nerve fiber
B. Formol corrosive - aka formol sublimate, belongs to
aldehyde fixative, preferably for post-mortem tissue
C. 10% Buffered Neutral Formal
D. Picric Acid - e
10% NBF
What type of metallic fixative is specifically used for
Wharton’s jelly and umbilical cord?
A. Lead fixative - contains large amounts of
mucopolysaccharide, esp. Acid mucopolysaccharide
B. Mercuric Chloride fixative - damages integrity of
tissue, preferably umbilical cord
C. Chromate Fixative - for carbohydates
D. Picrate Fixative - for histones and basic proteins
Lead Fixative
This section may be cut from tissues that have been
fixed and frozen with CO2 or for fresh tissues frozen
with a cryostat.
A. Celloidin section
B. Frozen section
C. Paraffin section
D. Gelatin section
E. NOTA
Frozen section
Sections are not left on the water bath for a long time
due to:
A. To avoid undue expansion of the tissue
B. To avoid distortion of tissue
C. Both
D. NOTA
To avoid undue expansion of the tissue and to avoid distortion of the tissue
BOTH
Brittle or hard tissues are caused by:
A. Prolonged fixation
B. Paraffin is too soft
C. Compress section
D. Sections are too thin
E. NOTA
Prolonged Fixation
Select which among the ff is a common component
of an aqueous media together with its correct use
A. Glycerol-prevent cracking
B. Glycerin-prevent drying
C. Gum Arabic- increase Refractive Index
D. Sugar-solidify medium
Glycerol
