Molecular Diagnostics Flashcards
What is the Central Dogma of molecular biology?
DNA is transcribed into RNA which is translated into protein.
What percent of DNA codes for protein?
10%
How does RNA differ from DNA
It is single stranded.
It contains a ribose sugar
It substitutes Uracil for Thymine.
How is mitochondrial DNA inherited?
Maternal inheritance
A translocation is an example of what type of mutation
structural
What is aneuploidy?
Abnormal number of chromosomes in a cell.
Whole chromosome losses are often associated with what?
Duplication of the remaining chromosome.
What change occurs in a nonsense mutation?
Changes a sense codon into a stop codon.
What is a silent mutation?
Alters a codon, but the same amino acid is expressed.
What type of mutation causes sickle cell disease?
Point mutation
What are the disadvantages of Southern blot?
Need for high molecular weight DNA
Labor intensive
long turnaround time
Which is NOT a sequence amplification technique?
a. PCR
b. transcription-mediated amplification
c. hybrid capture assay
d. ligase chain reaction
c. hybrid capture assay
What is the first step in Southern blot
DNA isolation
Which of the following is NOT a step in Southern blot?
a. digest DNA with restriction endonucleases
b. perform electrophoresis on dsDNA molecules
c. transfer DNA from gel to solid support
d. isolate high molecular weight DNA
b. DNA must be single stranded to hybridize to probe.
Which of the following is a characteristic of a loss of function mutation?
a. is frequently dominant
b. produces an entirely new trait
c. alters the protein structure and its functionality
d. causes a trait to appear in inappropriate tissues
c. alters the protein structure and its functionality
In Southern blot, which of the following does NOT affect the hybridization signal?
a. labeling efficiency of the probe
b. amount of tRNA transferred to the membrane
c. amount of genomic DNA
d. size of the probe
b. amount of tRNA transferred to the membrane
Which of the following is NOT a PCR reaction component?
a. deoxynucleoside triphosphates
b. ribonuclease
c. target DNA
d. DNA polymerase
b. ribonuclease
What are the steps of PCR in order?
denaturation - annealing - extension
Too little of any of the PCR reagents can lead to which of the following?
a. mispriming
b. thermodynamic infidelity
c. non-specific amplification
d. no amplification
d. no amplification
Which of the following types of PCR is used to analyze RNA?
a. Strand Displacement PCR (sdPCR)
b. Reverse transcriptase PCR (RT-PCR)
c. Transcription-mediated PCR (tmPCR)
d. Droplet Digital PCR (DDPCR)
b. RT-PCR
RT-PCR stands for what?
Reverse transcriptase PCR
What is the abbreviation for real-time PCR?
qPCR
(Quantitative PCR)
FISH is performed in situ on which of the following?
a. whole cells
b. nuclei
c. interphase/metaphase chromosomes
d. all of the above
d. all of the above
Which of the following is NOT a step in FISH?
a. denature chromosomes
b. denature probe and specimen DNA
c. hybridize probe to specimen DNA
d. detect monochromatic fluorescence
d.
FISH is used for which of the following applications?
a. identification and verification of chromosome translocations
b. gene mapping genomic research
c. rapid identification of species-specific chromosomes in somatic cell hybrids
d. all of the above
d.
Which of the following is a DNA sequencing method?
a. droplet digital PCR
b. ligase chain reaction
c. nucleic acid sequence based amplification
d. chain termination
d.
which of the following is a terminator in Sanger sequencing?
a. dideoxynucleoside triphosphate
b. ribonucleoside phosphate
c. deoxyribonucleoside diphosphate
d. deoxynucleoside monophosphate
a.
Which of the following is NOT a characteristic of next generation sequencing?
a. requires high quantities of purified DNA
b. sequences multiple genes simultaneously
c. provides high throughput comparatively
d. each DNA fragment is sequenced 10 to the x times.
a.
What is an analysis pipeline?
a. a number of steps to analyze next gen sequencing raw data
b. a series of analyzers linked together for increased efficiency
c. a data mining platform that was used in the human genome project
d. a diagnostic testing algorithm for a specific molecular disease
a. Steps used to analyze raw data
When referring to next generation sequencing, what are clusters?
a. groups of DNA templates in close spatial proximity generated by bead-based or solid phase amplification
b. groupings of similar sequences appearing in a particular region of a chromosome
c. frequently occurring SNPs that occur close to each other in a particular gene
d. groups of amino acid templates created by bead-based or by solid phase amplification
a.
What term defines the average number of times that a particular nucleotide is represented in a collection of raw sequences?
Depth
What is variant allele frequency?
The relative frequency of an allele at a particular locus in the population.
What myeoloproliferative disorder is BCR-ABL positive
Chronic myeloid leukemia (CML)
A JAK2 V617F gene mutation is seen in the majority of patients with which myeloproliferative disorder?
polycythemia vera
What gene mutation is a tyrosine kinase involved in signal transduction for multiple hematopoietic growth factors?
a. FLT3
b. JAK2
c. MYC
d. BCR-ABL
b. JAK2
Myeloid diseases result from genetic and epigenetic alterations that perturb which of the following processes?
a. proliferation
b. differentiation
c. self-renewal
d. all of the above
d. all of the above
Which of the following is an advantage of performing a myeloid NGS panel?
a. detection of large frameshift mutations (CALR/FLT3 genes)
b. correlation with morphologic and immunophenotypic data
c. ability to detect minimal residual disease in affected patients
d. 95% coverage of all target regions seen in myeloid disease
b. correlation with morphologic and immunophenotypic data
What is NOT a characteristic of PGXm (pharmacokinetic) precision medicine?
a. studies absorption and distribution of the drug in the body
b. links polymorphisms in drug disposition and effects
c. involves mostly mutations in disease causing genes
d. analyzes the metabolism and excretion of the drug
c. involves mostly mutations in disease causing genes
Which of the following is NOT a characteristic of PGXt (targeted therapy) pharmacogenetics?
a. germline versus somatic variants
b. polymorphisms affect whether the body rejects a drug
c. mostly mutations in disease causing genes
d. includes driver and passenger mutations
b. polymorphisms affect whether the body rejects a drug
Which of the following is an advantage of using NGS to analyze somatic mutations?
a. uses low quantities of extracted DNA
b. analyzes multiple genes simultaneously
c. analyzes multiple patient samples simultaneously
d. all of the above.
d. all of the above
The cfDNA correlates with which of the following?
a. tumor staging and prognosis
b. therapeutic selection
c. response rates
d. disease relapse
a. tumor staging and prognosis
The human genome contains approximately how many genes?
20,000-30,000
Which of the following is characteristic of a dominant disorder?
a. inheritance pattern is horizontal
b. affected individuals have a parent homozygous for the trait
c. unaffected individuals do not have affected children
d. males and females are affected differently
c. unaffected individuals do not have affected children
Which of the following is characteristic of a recessive disorder?
a. affected members of the family are usually siblings of the proband
b. affected individuals have affected parents
c. carrier parents have a 50% chance of having an affected child
d. inheritance pattern is vertical
a. affected members of the family are usually siblings of the proband.
Which of the following is NOT characteristic of alpha1-antitrypsin deficiency?
a. it causes early onset emphysema and chronic obstructive pulmonary disease
b. it is one of the most common childhood diseases requiring a liver transplant
c. it is one of the most common genetic disorders affecting Caucasians in the US
d. it is easily detected and 50% of the 25 million with one defective copy are diagnosed
d. it is easily detected and 50% of the 25 million with one defective copy are diagnosed
Which of the following is NOT characteristic of fragile X syndrome?
a. it is caused by a trinucleotide repeat expansion mutation
b. it was the first example of a single nucleotide deletion mutation
c. it is X-linked with incomplete penetrance
d. it is the most common form of inherited mental retardation and autism
b. it was the first example of a single nucleotide deletion mutation
When performing infectious disease molecular testing, which of the following extraction issues should be considered?
a. specimen type
b. target sequences
c. controls
d. all of the above
d. all of the above
For parvovirus B19 nucleic acid amplification, which of the following is NOT true?
a. target sequences include highly conserved sequences from NS-1 and VP-a
b. FDA-approved kits should be used routinely for regulatory compliance
c. test formats available include both qualitative and quantitative assays
d. acceptable samples include serum, bone marrow and amniotic fluid
b. FDA-approved kits should be used routinely for regulatory compliance
Which of the following is NOT an example of the clinical utility for parvovirus B19 molecular testing?
a. diagnosing fetal hydrops
b. diagnosing infection following seroconversion
c. determining the etiology of erythema infectiosum
d. detecting infection in transplant patients
b. diagnosing infection following seroconversion
Which of the following is NOT a characteristic of real-time PCR?
a. the results can estimate the starting quantity of target DNA in a sample
b. RT-PCR is often used synonymously with quantitative PCR
c. the results can be interpreted as detectable or undetectable amplification
d. it involves monitoring the fluorescent signal produced during every cycle
b. RT-PCR is often used synonymously with quantitative PCR
In the Delta Ct method of relative or comparative quantification, which of the following is true?
a. it assumes the same amount of template is present
b. the basic method corrects for efficiency
c. the basic method incorporates a normalizer
d. it assumes the amplification efficiency is 100%
a. it assumes the same amount of template is present
Which one of the following techniques fails to detect the presence of FUS-DDIT3 fusion gene in myxoid liposarcoma?
a. FISH
b. Karyotype
c. Allele-specific PCR
d. NGS
e. RT-PCR
c. Allele-specific PCR
The majority of the human genome is made up of coding sequences (genes). True or False
False
Which bases are purines?
Adenine, Guanine
Which bases are pyrimidines?
Cytosine, Thymine, Uracil
What is the difference between purines and pyrimidines?
Purines are double ring.
Pyrimidines are single ring.
How many hydrogen bonds between A and T?
2
How many hydrogen bonds between G and C?
3
Which ribose carbons carry the nitrogen base?
1’
Which ribose carbons participate in the phosphodiester bond?
3’ and 5’
Which carbon of deoxyribose carries the hydroxyl group?
3’
Which ribose carbon carries the phosphate group?
5’
How will methylation of cystosine bases 5’ to the gene affect gene expression?
Decrease
How will histone acetylation close to the gene affect gene expression?
Increase
How do siRNAs complementary to the gene transcript affect gene expression?
Decrease
What is the universal start codon?
AUG
Which amino acid does the start codon (AUG) code for?
Methionine
What are the stop codons?
UAA, UAG, UGA
How do you calculate the quantity of DNA?
Absorbance at 260 x 50 ug/mL x dilution factor
How do you calculate the quantity of RNA?
Absorbance at 260 x 40 ug/mL x dilution factor
How do you know the quality of DNA?
Take the 260/280 ratio.
Should be around 1.8
<1.6 contaminated with protein.
>2.0 contaminated with RNA
How do you know if an RNA isolation is good?
Take the 260/280 ratio
Should be above 2.0
How do you calculate melting temperature for short primer sequences
Tm = (4 degrees C x #GC pairs) + (2 degrees C x #AT pairs)
How does addition of formamide to affect stringency?
Increases
How does high salt concentration of hybridization solution affect stringency?
Lowers it
How does raising heat during hybridization affect stringency?
Increase
What causes primer dimers?
3’ complementarity in the primer sequences
Which control is run to detect contamination
Blank
How do you calculate the number of target sequences made in PCR?
2 to the nth power.
Where n is the number of cycles.
What does it mean if someone is heterozygous at the amelogenin locus? How many bands will be on the gel?
They are male. 2 bands
What does it mean if there is only one band after amplification and electrophoresis of amelogenin?
The specimen is from a female. (Homozygous)
Hardy-Weinberg equilibrium
Allele frequency in a population assuming a large population with random mating, no immigration, emigration, mutation or natural selection.
psquared+2pq +qsquared = 1.0
(p+q)(p+q)=1.0
Combined Paternity Index (CPI)
Multiply the paternity index of each locus tested
Probability of paternity
(CPIxprior odds)/(CPIxprior odds)+(1-prior odds)
Lab assumes 50/50 chance, so prior odds would be 0.5
One double-stranded DNA molecule with one or more non-complimentary bases.
Heteroduplex
How to enrich mRNA from a total RNA isolation
80-90% of RNA will be rRNA, only 2.5%-5% will be mRNA.
mRNA can be enriched by a column or beads with Thymine or Uracil that will bind to the polyA tail of mRNA.
rRNA subunits
28s and 18s
What are lncRNAs?
Long non-coding RNAs
Regulator of gene expression and chromatin structure.
What are siRNAs?
Short (or small) interfering RNAs
21-22 nt ds RNA
Regulator of gene expression
What are miRNAs?
MicroRNAs
17-27 nt
Regulator of gene expression
What is an oncomir
miRNA that acts as an oncogene or tumor suppressor
Target of antimer therapy
What is an operon?
A set of genes transcribed together on one polycistronic mRNA
How might a single mRNA produce more than one protein product?
Alternative splicing
What is imprinting?
Marking of selected regions of DNA, usually by methylation
What is a CpG island?
More than the expected # of CG dinucleotides often found 5’ to genes - targets for methylation
What is EDTA
Chelating agent - protects DNA
Component of TE Buffer (Tris EDTA)
Explain solid phase DNA isolation
Solid matrices to bind and hold DNA for washing
(e.g., spin columns)
Explain inorganic DNA isolation.
“Salting out”
Proteins precipitated with high salt concentration and low pH. Supernatant contains DNA which can be precipitated with EtOH
Explain organic DNA isolation
Phenol:Chloroform
upper aqueous phase contains DNA
What is the purpose of methylation?
Epigenetic regulation of gene expression
CG rich sequences
Main method of genomic imprinting
What is the size of the mitochondrial genome?
16,569 bp
How many genes are in the mitochondrial genome?
36
What types of diseases are due to mutations in mitochondrial genes encoding components of the oxidation-phosphorylation system?
neuropathies and myopathies
