Harr MLS Review Molecular Diagnostics

A FISH test is performed on a slide of peripheral blood leukocytes. The test uses a dual fusion probe, consisting of a Spectrum Green labeled probe to the BCR 22 q11.2 locus, and a Spectrum Orange labeled probe to ABL 9q34. What disease is this test for? A. Chronic myelogenous leukemia B. Multiple myeloma C. Bladder cancer D. Thyroid cancer

A. Although all of these cancers involve chromosomal ploidy or gene rearrangement that can be detected by FISH, the BCR/ABL translocation is found in >95% of CML and 25% of AML patients and in rare cases of chronic neutrophilic leukemia. There is a relationship between the type of leukemia and the portion of the BCR locus involved in the translocation. In CML, the major (M BCR) portion of the gene is involved, giving rise to a 210 dalton chimeric protein. In ALL, the minor (m BCR) portion is involved, giving rise to a 190 dalton chimeric protein. In CNL, an extended region beyond the M region, called the μ BCR region, is involved, giving rise to a 230 dalton chimeric protein. Also, variants in BCR/ABL exist, giving rise to different FISH patterns. Some variants are associated with essential thrombocythemia that occurs at disease onset in a small percentage of CML patients.

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

A. In PCR, the separation of dsDNA occurs by heating the sample to a temperature between 90°C and 94°C. This breaks the double bonds between the base pairs and is reversible by lowering the temperature. Alkali, high salt, and formamide also denature dsDNA, but they are not used in PCR because they would have to be removed and added with every cycle.

HLA typing can be done by which molecular method? A. PCR analysis using 96 well microtrays with allele or groups specific primers in each B. Restriction fragment length polymorphism testing C. Direct hybridization with WBCs on a peripheral blood film D. Fluorescent in situ hybridization reactions with peripheral blood lymphocytes

A. The DNA is extracted from peripheral blood leukocytes, added to the master mix, and an aliquot is transferred to each well of a 96-well plate. Each well contains a primer to a specific base sequence of one allele or allele group. Gel electrophoresis is performed after PCR to identify those wells that contain amplified products. Each well also contains a primer to a second nucleotide sequence, such as a region of the growth hormone gene that serves as a PCR internal control. Bands are stained with ethidium bromide and can be visualized by direct observation with a near ultraviolet light source.

What gene must be amplified in PCR to differentiate methicillin-resistant Staphylococcus aureus from methicillin-resistant coagulase-negative Staphylococcus? A. orfX B. mecA C. VanA D. iles-2

A. The mecA gene codes for resistance to methicillin in S. aureus and coagulase-negative Staphylococcus. In order to differentiate mecA-positive S. aureus from coagulase-negative Staphylococcus such as Staphylococcus epidermidis that are often present together in clinical specimens, the orfX gene specific for S. aureus is also amplified by multiplex PCR. A molecular beacon binds to the amplicon, causing fluorescence in real-time PCR, indicating the presence of both mecA and orfX products. The VanA gene codes for vancomycin resistance but is not found in S. aureus. The iles-2 gene codes for resistance to mupirocin in S. aureus.

p21 is a GTP binding protein produced by which oncogene? A. RET B. Ras C. HER-2/neu D. N-Myc

B. All of the genes are oncogenes. RAS is a group of three genes that produce GTP-binding proteins, which activate transcription by up-regulating the signal transduction pathway of the cell. RAS is implicated in lung, breast, colon, and other carcinomas. It is measured by RT-PCR, which quantifies the amount of mRNA present in the malignant cells.

Which oncogene is involved in the etiology of Burkitt's lymphoma? A. ABL B. Myc C. Ras D. HER/neu

B. Burkitt's lymphoma is associated with a translocation involving the long arm of chromosome (8 on which the c-myc gene is located) with one of three immunoglobulin genes. The translocation most often involves the IgH gene on chromosome 14. The result is a hybrid mRNA that produces the c-myc protein whenever the immunoglobulin gene is transcribed. The c-myc protein is an activator of genes involved in mitosis.

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

B. In PCR, several target sequences can be tested for simultaneously using multiple primers (multiplex PCR). Several methods exist for detection and quantitation of possible PCR products. The traditional method is Southern blotting, in which fluorescent- labeled probes to each template hybridize with their respective product after PCR. Alternatively, the primers can be labeled with different fluorescent dyes. These can be detected after PCR by capillary electrophoresis using laser-induced fluorescence.

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

B. PCR primers are small oligonucleotides, usually 12 to 36 bases, complementary to the base sequence at the 3 ́ end of the target DNA. Two primers are used, one to the sense strand of DNA (the strand containing the gene) and the other to its complement (the antisense strand). Primers for PCR are made only for the 3 ́ end of each target sequence because the DNA polymerase that extends the primer does so only by addition of bases in the 5 ́ → 3 ́ direction.

How are cases of Duchenne's muscular dystrophy not detected by PCR usually confirmed? A. DNA sequencing B. Linkage analysis C. Macroarray analysis D. Dystrophin protein staining

B. The majority of gene deletions associated with Duchenne's muscular dystrophy is detected by PCR using multiple primers (multiplex PCR). The others are usually detected by indirect gene analysis. An alternative to testing for these mutations is linkage analysis. This process follows other genetic markers located near the disease gene so that crossing over is improbable. Linkage analysis for an X-linked disease or an autosomal recessive disease such as cystic fibrosis requires DNA from at least one affected family member. However, linkage analysis for an autosomal dominant disease such as Huntington's disease requires DNA from at least two family members. Next generation gene sequencing (several technologies that are alternatives to Sanger sequencing) have been used to identify deletions of the dystrophin gene that cannot be detected by PCR.

Inheritance of BRCA1 or BRCA2 mutations increases the risk of breast and ovarian cancer by which mechanism? A. Oncogene production B. Transcription signaling by the mutant protein C. Deficient tumor suppressor function D. Chimeric protein production

C. BRCA1 and BRCA2 are mutations of genes that produce tumor suppressor proteins. These down-regulate cell signaling events that lead to cell division. The mutations are inherited as autosomal dominant traits and are associated with > 85% lifetime risk at age 70 of developing breast cancer if one is found in a person with a positive family history.

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

C. Because heat is used to denature dsDNA with every cycle of PCR, the polymerase used must be heat stable. Taq polymerase is obtained from Thermus aquaticus, a bacterium that lives in the hot springs of Yellowstone National Park. It retains its activity even after repeated heating at 95°C. The optimal temperature for extension by Taq is 72°C. A typical PCR cycle involves heating to 94°C for denaturation, cooling to 64°C for annealing, and heating to 72°C for extension.

Which statement best describes real-time PCR testing for Mycobacterium tuberculosis? A. The test is positive only in cases of smear-positive and culture-positive infections B. The test has a sensitivity of >99% on all specimen types when compared to culture C. The test can detect 85%-90% of smear-negative, culture-positive infections D. The test sensitivity is near 100% but specificity is approximately 80%

C. PCR detection is dependent on having at least a minimal number of organisms present in the specimen, and sensitivity is 90% or lower when the specimen is AFB smear negative but culture positive. Specificity of PCR is approximately 98%.

The majority of cases of Duchenne's muscular dystrophy are caused by which type of genetic damage? A. Point mutation B. Insertion C. Deletion D. Trinucleotide repeats

C. The dystrophin gene is approximately 2.5 million bases and has extensive sites at which both large and small deletions, insertions, and point mutations can occur. Approximately 60% of cases are caused by deletions that can be detected by the absence of one or more PCR products produced by the normal gene. The remaining 40% can be caused by microdeletions, point mutations, or insertions that are not usually detected by available primer sets.

Hereditary hemochromatosis is the result of which type of mutation? A. Nonsense mutation B. Microdeletion C. Translocation D. Single nucleotide substitution

D. Hereditary hemochromatosis is an autosomal recessive disease with a frequency as high as 0.5% in the White population. The mutation occurs in the HFE gene on chromosome 6 and involves a single base that results in tyrosine substituting for cysteine in the HFE protein. The HFE protein down-regulates iron absorption. The mutant protein usually increases iron absorption by at least 100%. Homozygous HFE mutation (C28Y) accounts for approximately 80% of hereditary hemochromatosis. The remaining cases are caused by a single-point mutation at position 63 on the protein (H63D), which produces a milder increase in iron absorption. Genotype is determined by PCR using specific oligonucleotide probes to identify the products.

Which of the following parameters are used to gate cells processed by the flow cytometer? A. Font surface fluorescence versus incident laser intensity B. Forward light scatter versus side scatter C. The ratio of light emitted at two different wavelengths D. Impedance amplitude versus background conductance

B. The gated population is selected by evaluating the scatterplot of forward light scattering (x axis) and right angular or side scatter (y axis). Cells falling within the specified limits are counted. For example, monocytes can be differentiated from neutrophils because the former have greater forward scatter and less side scatter.

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

B. The threshold cycle (Ct) is the point of maximum curvature in the signal plot of fluorescence versus PCR cycle. It is the number of cycles needed before PCR product is detected. The Ct is determined for each DNA standard and is plotted against the log of the concentration. This gives a straight line with a negative slope because the Ct is inversely related to the concentration of the PCR product. A standard curve is also required for a reference gene. The reference gene is one that will display the same amplification from sample to sample. The signal for the target is divided by the reference signal to correct the measurement for error caused by variable rates of target amplification. This occurs because the samples contain variable amounts of DNA to start with.

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

C. In real-time PCR, the melting temperature (Tm) corresponds to the temperature at which half of the DNA product separates into single strands. When the negative first derivative (-∆F/∆T) is plotted against temperature, the melting peak for the PCR product is produced. When more than a single melting peak occurs, there is more than a single PCR product. Thus, melting temperature analysis can identify situations where an unexpected product or a contaminant may be present.

Which method is most sensitive for detection of viral meningitis? A. Viral culture B. CSF WBC count C. Specific antibody testing of CSF for viral antigens D. Real-time RT-PCR

D. Enterovirus is the most common cause of viral meningitis, accounting for more than 85% of cases. Viral culture is positive in 50%-70%, while the sensitivity of real-time PCR is above 95%. Enterovirus strains show significant homology at the 5' end, making it possible to detect several different enterovirus serotypes using a single primer pair. The PCR reaction is not inhibited by antiviral therapy and can be done in hours versus days for viral culture.

Which mechanism is responsible for retinoblastoma? A. Mutation of a tumor suppressor gene B. Mutation of a tyrosine kinase gene C. Activation of an oncogene D. Deletion of a gene encoding a GTPase activator

A. A mutation or deletion of a tumor suppressor gene such as p53, p14, or RB1 (the retinoblastoma gene) causes loss of a protein that inhibits mitosis and is associated with an increased risk of malignancy. Mutations of p53 occur frequently in several cancers, including lung, breast, liver, and colon cancer. RB1 mutations are associated primarily with retinoblastoma, a tumor of the retina occurring in young children. Although they may be inherited, mutations usually arise in somatic cells. Mutations that produce more active proteins with tyrosine kinase activity such as HER-2/neu are oncogenic because they stimulate the signal transduction pathway for mitosis. Likewise, a deletion of a GTPase activator is also oncogenic, since it permits higher levels of intracellular GTP, which is involved in the same pathway.

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

A. A plasmid is a piece of circular double-stranded DNA located in the cytoplasm of a bacterium. Although not attached to a chromosome, the plasmid is replicated like chromosomal DNA. The plasmid is cut with the restriction endonuclease that is used to isolate the DNA fragment containing the gene of interest. The fragment anneals to the sticky ends of the plasmid DNA, and the cut is repaired by DNA ligase. The recombinant plasmid is added to a culture of bacteria that is disrupted to promote the uptake of plasmid DNA. Commercially available plasmids have promoter and reporter genes such as lac and lacZ that produce β-galactosidase. These can be used to identify colonies with successful recombinants. They also carry antibiotic resistance genes that allow the recombinants to be purified. Culture of the recombinant bacteria results in large amounts of the gene, which can be harvested using the restriction enzyme, denatured, and labeled to make the probe.

Which statement best describes the relationship between HLA DNA typing and serological haplotypes? A. One or two bands are seen for each locus correlating to reactivity with a specific antigen or group of antigens B. HLA alleles cannot be related to HLA antigens because antisera specificity is unrelated to genetic polymorphism C. A single antibody specificity always corresponds to a single allele D. Not all HLA genes produce antigens recognized by antibodies

A. Antibodies to HLA antigens recognize determinants that may be shared by several polymorphisms. However, it is possible to correlate primer specificities to gene products that react with commercial HLA typing seras. For example, DR103 correlates with the primer recognizing DRB1*0103. On the other hand, alleles DRB3*010101-10, DRB3*0101-14, and DRB3*030101-03 will all react with antisera to DR52.

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)

A. Approximately 80% of polymorphisms result from single nucleotide substitutions and are called single nucleotide polymorphisms. Some SNPs are silent, whereas others cause a change in the codon within the gene. VNTRs, STRs, and SINES refer to polymorphisms involving differences in the length of as opposed to the sequence of bases. These are specific base sequences that occur throughout the genome that are repeated at a particular locus. The number of times the sequence repeats is an inherited trait. For example, the sequence AATG is a repeat that occurs within the tyrosine hydroxylase gene on chromosome 11. The sequence can repeat 3 to 14 times, resulting in 12 different alleles. Someone who inherits allele 6 (AATG repeats six times) will have a DNA molecule that is four base pairs longer than someone who inherits allele 5 (AATG repeats five times). This locus, called TH01, is used in forensic and parentage testing to establish identity.

A cell population is positive for surface markers CD45, CD3, CD4, and Tdt. Which type of leukocytes are these? A. Lymphocytes B. Granulocytes C. Monocytes D. Early myeloid precursors

A. CD45 is a panleukocyte marker and reacts with all white blood cells and precursors. CD3, CD4, and Tdt are markers for T lymphocytes. Typically, a panel of 12 or more antibodies is used to characterize the lineage and maturity of a cell population. The abbreviation CD stands for cluster of differentiation. Monoclonal antibodies with the same CD number recognize the same marker, although the specific moiety they react with may be different.

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

A. Capillary electrophoresis (CE) is a method commonly used to separate DNA fragments. Unlike conventional electrophoresis, a stationary support such as agarose is not used. Instead, a small-bore open tubular column is immersed in buffer solution at its ends and subjected to an electric field. Molecules such as proteins and DNA are injected by application of either pressure or high voltage (electrokinetic transfer). The negative nature of the glass capillary attracts cations that are pulled to the cathode when the voltage is applied. This creates an electro-osmotic force (EOF) that draws water and other molecules toward the cathode. An ultraviolet light detector or laser-induced fluorescence detector is located near the cathode and detects the molecules as they migrate. At an alkaline pH, DNA and protein molecules are negatively charged but are pulled toward the cathode by EOF at a rate inversely proportional to their size. CE columns can be coated with a gel such as acrylamide or a polymer that neutralizes the EOF, so that the DNA molecules are drawn toward the anode at a rate inversely related to the molecular size. DNA molecules such as PCR products of 100 to 1,000 base pairs can be detected with a band resolution as high as 1-2 base pairs and a sensitivity of approximately 1 ng/mL DNA. Such high resolution is possible because very high voltage can be used, since the heat produced is lost through the capillary wall.

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

A. Concentration is exponentially related to fluorescence at the start of the PCR reaction, but beyond the early exponential phase, the relationship does not hold. The cycle threshold is in the exponential phase of amplification, which is why the Ct is used for quantification. Since in theory, each cycle doubles the amount of PCR product, two samples that differ in Ct by four cycles differ in PCR product by 16-fold. However this assumes 100% efficiency in the PCR reaction and does not take into account differences in the amount of starting material. A commonly used formula to estimate PCR product is the ∆∆CT method. The sample PCR product is normalized by subtracting the PCR product of a reference gene (such as actin). In addition, a control cell is also measured and its product is subtracted from the test sample after subtracting the signal for the same reference gene. Relative concentration = 2^(-∆∆CT), where the ∆∆CT = ∆Ct sample minus ∆Ct control.

Which is the most common method used for parentage testing in the United States? A. Short tandem repeat analysis B. Nuclear DNA sequencing C. HLA DNA typing D. Mitochondrial DNA sequencing

A. DNA testing is the primary method of determining parentage because it is 100% accurate in exclusion and > 99.9 % accurate for inclusion of parentage. DNA testing is at least 10-fold more conclusive than the combination of HLA, blood group, and protein markers, and DNA samples can be tested prenatally, neonatally, and postmortem. Testing is performed on nuclear DNA because mitochondrial DNA is inherited exclusively from the mother. Rather than testing for base sequence variations within genes, DNA is tested for length polymorphisms. These are short base sequences within the introns that repeat. The number of times the sequence repeats is inherited as a trait. Short tandem repeats (STRs) are oligonucleotide sequences of four or five base pairs. Usually, 12 STR loci are amplified by PCR using specific oligonucleotide primers labeled with fluorescent dyes. The products are detected by capillary electrophoresis. The size of the fragments and their fluorescence determine which alleles are present.

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

A. Each phosphoric acid subunit within a phosphodiester bond to adjacent deoxyribose molecules has a single negative charge at an alkaline pH. Since the charge is distributed evenly, smaller fragments move more rapidly through the gel. When suspended in an alkaline buffer (pH 8) such as tris-borate-EDTA (TBE) or tris-acetate-EDTA (TAE), the DNA fragments migrate toward the anode at a rate that is inversely proportional to the log10 of molecular size. If the distance traveled is plotted against the log of molecular weight, the plot will be a straight line with a negative slope because the larger the molecule, the more slowly it moves through the pores of the gel. The plot can be calibrated with a DNA size ladder, and the molecular weight of DNA fragments can be determined from the calibration curve.

In general, which statement best characterizes the relationship between white blood cells and light scattering in flow cytometry? A. Forward scatter is related to cell size and side scatter to granularity B. Forward scatter is related to nuclear density and side scatter to size C. Forward scatter is inversely related to size and side scatter is directly related to size D. Forward scatter is related to shape and side scatter to size

A. Forward scatter of light from a laser directed through the aperture of the cytometer is directly related to cell size. Right angular scatter (side scatter) is dependent upon the number of granules inside the cytoplasm. For example, small lymphocytes that are agranular have the lowest forward and side scatter and are easily identified as the cluster of cells closest to the bottom and left of the scatterplot.

In flow cytometry, the term "gating" refers to: A. Selection of a subpopulation of cells to count B. Determining the fluorescent emission spectrum of cells of interest C. Interference caused by binding of more than a single antibody D. Selecting the appropriate counting aperture

A. In flow cytometry, cells can be divided into subpopulations based upon their light-scattering properties. Cells to be interrogated by the laser(s) are selected by identifying the area in which they appear on a scatterplot.

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

A. In real-time PCR, the fluorescence of the reporter probe is proportional to the concentration of PCR products. For quantitation of PCR products, a well factor and background fluorescence must be determined. Well factor values are analogous to cuvette blanks. They are used to correct the measurements from each well so that the same concentration of fluorescent dye gives the same signal intensity regardless of the well. The threshold is the lowest signal that indicates the presence of product. It can be calculated manually from a real-time amplification curve by finding the average standard deviation of the fluorescent signal (RFU) from cycles 2-10. This is multiplied by 10 to give the threshold value in RFUs.

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

A. Master mix solutions must contain all of the reagents needed to generate new dsDNA. This includes DNA polymerase, the enzyme needed to replicate the target sequence, primers to initiate replication, magnesium (a polymerase cofactor), buffers to maintain pH, and deoxyribonucleotide triphosphates that are the substrates for DNA polymerase (adenosine triphosphate, guanosine triphosphate, thymidine triphosphate, and cytosine triphosphate).

What method is used to identify maternal cell contamination in amniocentesis and chorionic villus samples (CVS)? A. STR analysis B. FISH C. Microarray analysis D. MicroRNA (MiRNA) analysis

A. Maternal cell contamination can result in misinterpretation when performing genetic testing directly on uncultured CVS or amniotic fluid cells. FISH can identify maternal cell contamination if the fetus is male but not female. STR analysis using 5 loci can detect maternal cell contamination as little as 1%. A level of maternal contamination below 1% does not guarantee accuracy, but misinterpretation due to maternal contamination is unlikely. Negative genetic tests can be reported, but positive results should be confirmed using cultured cells. Maternal contamination is more common from CVS than amniotic fluid samples. MiRNAs are small RNA molecules that bind to mRNA and block their translation. There are about 500 miRNAs in human cells and their expression has been used to identify the tissue of tumor origin.

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

A. One method of preventing PCR products from previous assays from contaminating a sample or test in progress is to substitute the RNA base uracil for thymine in the PCR products. Taq polymerase will insert deoxyuridine phosphate instead of deoxythymidine phosphate during the primer extension phase of each cycle. The enzyme uracil N-glycosylase is added to the master mix along with deoxyuridine triphosphate, which replaces deoxythymidine triphosphate. Prior to the first denaturation, the enzyme hydrolyzes the bond between uracil and deoxyribose. When the sample is heated to separate the strands, the enzyme becomes denatured and any contaminating PCR products fragment into small oligonucleotides that cannot be replicated.

Which formula predicts the number of PCR products that can be produced? A. 2^n where n is the number of cycles B. N^4 where N is the number of cycles C. p^2 + 2pq + q^2 = 1 where p and q are the number of primers D. N^2/2 where N is the number of cycles

A. PCR has the potential to double the quantity of PCR products with every cycle. Therefore 2^n predicts the number of PCR products that can be produced from n cycles. For example, if 30 cycles are programmed, then 2^30 predicts slightly over 1 billion PCR products. The formula p^2 + 2pq + q^2 = 1 describes the distribution of a two-allele gene in a population.

Polymorphisms of the cytochrome p450 genes are important in identifying which condition? A. Poor drug metabolism B. Risk for primary biliary cirrhosis C. Progression of hepatitis C to hepatic cirrhosis D. Parentage in cases where HLA results are inconclusive

A. Pharmacogenetics (sometimes called pharmacogenomics) is the study of the role inheritance plays in the metabolism of drugs. Individual differences in drug metabolism can be attributed in part to polymorphisms in the genes coding for enzymes comprising the cytochrome p450 system. Of the more than 100 CYP genes, seven are principally involved in drug metabolism. Of these, CYP2D6, CYP2C9, CYP2C19, and CYP2A6 are polymorphic genes that account for metabolism of approximately 40% of drugs. Phenotypical expression varies with the locus involved. For the CYP2C19 locus that metabolizes several dozen drugs—including some tricyclic antidepressants, antiepileptics, and acid reflux inhibitors—persons who inherit one copy of the wild-type gene metabolize normally, whereas homozygotes or double heterozygotes for any of the seven polymorphisms metabolize poorly. For CYP2D6—which metabolizes tricyclic antidepressants, antipsychotics, antihypertensives, and several other drugs—heterozygotes with one wild-type gene have intermediate and those with no wild-type gene have poor drug metabolism. Persons with poor metabolic efficiency are at a greater risk of drug toxicity. On the other hand, their response to some antibiotics may be more positive.

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

A. Quantitative PCR can be used to measure viral load and gene expression. However, the PCR process is associated with a high run-to-run variance that can be reduced by simultaneously measuring the PCR products of an internal standard of known concentration (molecules per PCR). For example, in competitive PCR, a DNA template having the same primer binding region but that is shorter than the native DNA is added to each sample. The signal used to determine concentration is derived from the ratio of the native DNA product to the competitive template product. This value is compared to the signal generated by adding a known amount of DNA from a reference gene (internal standard) and is reported as copies per milliliter or copies per molecule of reference gene. Some quantitative PCR methods use external standards. However, an advantage of the internal standard method is that the calibrator is subject to the same influences as the target DNA by being mixed with DNA from the patient's sample.

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

A. Southern blot hybridization is a method commonly used to detect disease genes in both PCR products and RFLP testing. The DNA fragments are electrophoresed, and the DNA bands are transferred by suction to a nylon or nitrocellulose membrane. The bands are immobilized and denatured on the membrane, and a solution containing the labeled probe is added. Hybridization is the binding of the complementary base sequence of the probe to the target sequence. This process is highly dependent upon temperature, ionic strength,and the presence of reagents in the hybridization solution that influence stringency (the degree of exactness of base pairing). A Northern blot test follows the same process, except that the sample is RNA. In a Western blot test, the sample is a mixture of proteins, and the probes used are (labeled) antibodies to the proteins of interest. A dot blot is a hybridization method in which samples of DNA are placed directly on the nitrocellulose membrane as a circular spot (or bar in the case of a slot blot), followed by the hybridization process.

Which of the following thalassemias can be detected by PCR followed by blotting with a single specific oligonucleotide probe? A. α-Thalassemia B. Hemoglobin S/β-thalassemia C. β-Thalassemia D. Hemoglobin S/β-thalassemia

A. α-Thalassemia carriers have a full or partial deletion of one or two of their four globin genes. Genotyping can determine whether two deletions are cis or trans and is performed by PCR using primers that are specific for the four most common deletions. β-Thalassemia may be caused by single base substitutions, deletions, or mutations in the flanking regions of the β-gene. Over 200 different mutations have been described, and 20 are relatively common. Microarray analysis is required to detect these.

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

B. A nonsense mutation occurs when a nucleotide substitution within a codon changes the code from that for an amino acid to a stop sequence. For example, a change from TTC to GTC changes the mRNA transcript from AAG to UAG. AAG codes for lysine and UAG is a stop codon; therefore, instead of lysine being added to the protein during translation, protein synthesis is terminated. In the reverse situation, the point mutation changes a termination codon into one for an amino acid and a longer protein is produced. A missense mutation occurs when a base substitution alters the codon so that a different amino acid is inserted during translation. A frame shift mutation occurs when there is a deletion or insertion of more or less than three bases. This changes the triplet order, altering the amino acid sequence of the protein.

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

B. An array is an organized arrangement of known molecules (either DNA or proteins for proteomic array analysis). DNA arrays are used primarily for studying gene expression and single nucleotide polymorphisms. Commercially prepared arrays use short synthetic oligonucleotides (12-36 bases) of single-stranded DNA immobilized onto a substrate, usually a glass or a silicon chip. These are usually called the targets, and a single array can contain hundreds to many thousands of targets. The sample DNA is usually derived by RT-PCR of test cells. This produces single-stranded complementary DNA (cDNA) representative of active genes within the cells. These are labeled with one or two fluorescent dyes and therefore are usually called probes. However, some commercial systems refer to the immobilized (array) DNA as the probe and the labeled DNA as the target.

How can cell proliferation be explained by the BCR/ABL gene rearrangement that occurs in the 9:22 translocation that causes the Ph1 chromosome of CML? A. It causes underexpression of p53 B. A hybrid protein is made that up-regulates the cell cycle C. Translocation induces a point mutation in the ABL oncogene D. ABL activates p23

B. Cancers are caused by genetic damage to cells that disrupt the cell cycle. Cell proliferation can be induced by under-expression of genes with tumor suppressor properties (e.g., p53) or overexpression of oncogenes (e.g., p21) that increases cell signaling, transcription, and mitosis. In CML, translocation of the ABL oncogene from chromosome 9 to the 3 ́ end of the BCR (breakpoint cluster region or area where recombination occurs) of chromosome 22 results in production of a hybrid BCR/ABL mRNA. This produces a chimeric protein with increased tyrosine kinase activity, causing the cell to enter G1. FISH can be used to identify cells with the BCR/ABL translocation. DNA probes specific for ABL and BCR are labeled with two different fluorescent dyes. In normal cells, each dye produces two colored spots (e.g., red and green) on chromosome pairs 9 and 22. If a BCR/ABL translocation is present, the probes bind next to each other, producing a spot of a different color (e.g., yellow).

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

B. Exons are the components of genes that determine the amino acid sequence of the protein synthesized. Exons are separated by noncoding regions called introns that are transcribed and later removed from mRNA before translation. Promoters are sequences located near the gene at the 5 ́ end and facilitate binding of proteins that increase transcription. A TATA box is an oligonucleotide sequence often found in the promoter region. The AT base pairs have two hydrogen bonds that separate more easily than CG bonds, thus creating a point where the double helix is easier to open.

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

B. FISH is used to detect abnormalities of chromosomes in cells and tissues by facilitating the direct attachment of a fluorescent-labeled oligonucleotide probe or probes to the chromosome. Hybridization of the oligonucleotide probe requires treatment of the cells with proteinase K and other agents such as nonionic detergent to increase permeability. Prehybridization may be required to decrease background fluorescence. Denaturation requires controlled temperatures at or near the melting point and the addition of a hybridization solution. This usually contains formamide, sodium chloride and sodium citrate, and EDTA to weaken the hydrogen bonds of the dsDNA target. Hybridization of the fluorescent-labeled probe(s) to the chromosomal DNA also requires controlled temperature incubation. After incubating with the cells, any unattached probe is removed by washing, and the cells are examined with a fluorescent microscope containing the appropriate filters to transmit the excited light from the specific probe(s).

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

B. Fluorescent or enzyme labels can be attached to probes by nick translation. A DNase is used to cut the probe at a few phosphodiester linkages. PolI repairs the nicks by removing nucleotides from the 3 ́ end and replacing them with labeled nucleotides at the 5' end of the nick. Alternatively, a primer containing a labeled nucleotide can be used to make copies of the probe by DNA amplification (PCR). A common label used for probes consists of biotin conjugated to the 5' end of the probe. After hybridization, streptavidin conjugated to an enzyme such as alkaline phosphatase is added. Streptavidin strongly binds to biotin, forming an enzyme-labeled complex with the DNA. After washing to remove unbound streptavidin, a colorimetric, fluorescent, or chemiluminescent substrate is added.

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

B. Protein microarray analysis uses immobilized bait to isolate proteins from serum, body fluids, or cell lysates. The array may contain antibodies, antigens, receptor molecules, or protein binding ligands (e.g., drugs). The proteins can be identified by fluorescent- or enzyme-labeled probes and can be analyzed by mass spectroscopy to produce a fingerprint of the proteins isolated on the array. This can be compared to a learning set, a combination of proteins that is associated with a specific disease such as ovarian cancer. If the pattern falls within specified parameters determined by the learning set, then cancer is identified. Analysis is based upon determining the time required for each protein to move through a mass filter. Two related instrument principles are used, matrix assisted laser desorption ionization—time of flight mass spectrometry (MALDI-TOF), and surface enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF). Both use a laser to ionize the proteins and a mass filter to separate them based upon their mass/charge ratio. Since protein expression of cancer cells is altered before morphology changes, the analysis of protein patterns of serum and suspected cells provides an opportunity for diagnosis at an early stage of progression or at a premalignant state.

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

B. Some samples may contain inhibitors of the PCR reaction. For example, a sample in which DNA was extracted using a cation chelator to prevent DNA degradation may be contaminated with residual chelating reagent. Since DNA polymerase requires Mg+2, this will inhibit amplicon production. An internal control can identify this problem. The sample is mixed with the internal control, a DNA molecule with the same primer binding region. The internal control should always be amplified, but the product can be distinguished from the target amplicons. Failure of a sample to demonstrate the internal control product in an assay where positive and negative control reactions are valid indicates the presence of an inhibitor in the sample.

Which method of DNA analysis is used most often to detect the hemoglobin S gene? A. FISH B. PCR followed by RFLP C. Cytogenetic analysis of chromosome 11 D. Labeled probe painting of chromosome 11

B. The β-globin gene is located near the end of the short arm on chromosome 11 and consists of three exons and two introns constituting 1,600 base pairs. The substitution of valine for glutamic acid at position 6 of the protein is the result of a single-point mutation at position 6(A3) in exon 1, in which GAG is replaced by GTG. In hemoglobin C, the same codon is mutated but the substitution involves the preceding base at the 5 ́ end (GAG is changed to AAG). The hemoglobin S mutation alters the restriction site for MstII, preventing the enzyme from cutting the DNA. This causes production of a fragment that is 200 base pairs longer than seen for the normal β-gene. Most commonly, PCR is used to amplify a portion of the exon containing the S mutation, and MstII is used to digest the PCR product. Heterozygotes produce one normal and one longer band, whereas homozygotes produce a single band that is 200 base pairs longer than the normal amplicon. Alternatively, PCR is performed followed by Southern blotting, using specific oligonucleotide probes for hemoglobin A and S.

In order to prove exclusion in DNA paternity testing, why must two genes be identified that must come from the biological father and did not? A. A single exclusion can result from laboratory error B. A single exclusion can result from germ line mutation within one locus being tested C. The biological father may be a blood relative to the alleged father D. The biological mother may be different than the purported mother

B. Two exclusions are needed rather than one to be 100% certain of nonpaternity because of the rare possibility of a mutation having occurred in one of the loci being tested. Loci used for DNA testing are sufficiently polymorphic that the mother's sample is not necessary to determine paternity. Exclusion is based on the premise that the biological father must have at least one allele in common with the child at each locus.

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

B. When messenger RNA is transcribed, the enzyme polyA polymerase adds 50 to 250 adenine bases to the 3 ́ end of the molecule. This polyA tail protects the mRNA from enzymatic degradation and promotes its binding to the ribosome. Since almost all eukaryotic mRNA has a polyA tail, oligo dT primers are used to initiate reverse transcription, making cDNA copies of the mRNA, and oligo dA primers are used to initiate amplification of the cDNA product.

Which method of analysis is considered the most reliable means of detecting mutations of BRCA, p53, BRAF, and other genes linked to cancer? A. FISH B. Immunohistochemistry C. Sequencing D. STR analysis

C. Genes such as BRCA1 and BRCA2 are large and thousands of point mutations have been identified, making it impossible to perform an assay using DNA probes. When many mutations of a gene are possible, the most widely used detection method is sequencing. Next generation sequencing has made clinical applications affordable. The process involves creating a library of templates, copying them by PCR, separating the copied strands, and determining their base order by PCR using a reversible dye termination reaction. When a fluorescent-labeled nucleotide is added a laser determines its emission and the base is identified. The blocking group and fluorescent dye are removed and the process repeated for the next base added.

Which method is most useful for confirmation that a culture isolate is Group B streptococcus? A. Southern blotting B. Polymerase chain reaction C. Direct hybridization D. Probe capture assay

C. In direct hybridization, a specific labeled probe reacts directly with the sample. Since a colony or pure broth culture of a primary isolate represents the progeny of a single bacterium, there is no need for the use of Southern blotting. The quantity of DNA available for testing is sufficient, so that amplification methods such as PCR or probe capture hybridization are unnecessary. The colony or broth isolate is lysed, and a hybridization solution is used to promote denaturation. The sample is heated above the melting temperature, and a DNA probe is added that hybridizes with bacterial DNA or ribosomal RNA. The probe is conjugated to a chemiluminescent label. A reagent is added to neutralize the unbound probe, and H2O2 and NaOH are added to cause chemiluminescence. The signal is read in a luminometer and compared to a cutoff value. Such tests take approximately 1 hour to perform and most are 99%-100% sensitive and specific.

Fluorescent dyes most commonly conjugated to antibodies used in flow cytometry are: A. Fluorescein isothiocyanate and Texas red B. Calcofluor white and Texas red C. Phycoerythrin and fluorescein isothiocyanate D. Acridine orange and rhodamine

C. In flow cytometry, cells are mixed with a panel of specific antibodies that bind to surface antigens that characterize their lineage and maturation state. The antibodies are conjugated to fluorescent dyes that are excited by the laser. If light of the characteristic wavelength emitted by the fluorescent label is detected, then the cell bound the labeled antibody and is positive for the respective antigen. The two most frequently used dyes are fluorescein isothiocyanate (FITC) and phycoerythrin (PE). Since they emit green and red light, respectively, they can be differentiated in the same sample, allowing two antibodies to be tested simultaneously. Using more dyes such as PE-Texas Red allows for the simultaneous measurement of more markers. For example, different fluorescent dyes can be attached to latex beads in different proportions so that up to 100 combinations can be discriminated by the optics. This allows 100 different markers to be measured in the same sample simultaneously. Flow cytometry is used to measure specific plasma proteins and antibodies using fluorescent antibody-coated beads.

How can cell proliferation be explained by the BCL 2 translocation t(14;18) that occurs in up to 90% of persons with follicular B-cell lymphoma? A. p53 is underexpressed B. A hybrid protein is made that up-regulates the cell cycle C. Transcription of the BCL 2 oncogene is increased by the translocation D. The BCL 2 gene joins with the p21 gene, making it inactive

C. In follicular B-cell lymphoma, relocation of the BCL oncogene next to the gene for the immunoglobulin heavy chain (IgH) occurs. The BCL oncogene product is a protein that inhibits apoptosis. When the cell transcribes the IgH gene, it produces the BCL 2 protein as well, which protects the cell from apoptosis. This translocation occurs in all cases of follicular B-cell lymphoma and can be identified using FISH with fluorescent-labeled DNA probes to IgH and BCL 2 genes.

In situ hybridization (ISH) tests for human papilloma virus (HPV) using cervical smears differ from immunochemical staining of tissue in which regard? A. ISH has lower analytical sensitivity B. ISH has lower analytical specificity C. ISH differentiates subtypes more easily D. ISH differentiates cervical neoplasia from genital warts

C. In situ hybridization using probes that anneal with specific subtypes of HPV are able to distinguish the subtype of virus most commonly responsible for sexually transmitted warts and associated with neoplasia. Positive reactions can be detected by light microscopy using probes conjugated to biotin. After the hybridization reaction, the slides are washed to remove the unbound probe, and streptavidin conjugated to horseradish peroxidase is added. Addition of hydrogen peroxide and aminoethylcarbazole results in the formation of a reddish-brown precipitate. Sensitivity is approximately 88% and specificity 99%, which is higher than for histochemical immunoperoxidase staining. HPV is present in normal-appearing cells as well as those demonstrating intraepithelial neoplastic lesions. However, persons testing positive for HPV types associated with cervical cancer such as type 16 are at higher risk for the disease.

What is the clinical significance of K-ras testing? A. K-Ras mutations make tumor cells more susceptible to chemotherapy B. K-Ras is a tumor suppressor gene and mutations are associated with increased lifetime risk of malignancy C. K-Ras mutations result in treatment resistance to growth factor receptor inhibitors D. K-Ras is used to identify the tissue of origin

C. K-Ras is a proto-oncogene that makes a GTP binding protein. When the protein is bound to GTP, it initiates a cascade of phosphorylation reactions leading to transcription. K-Ras is activated when an epidermal growth factor binds to the epidermal growth factor receptor (EGFR). EGFR is overexpressed in several cancers including colorectal, lung, and pancreatic cancer. These can be treated with EGFR inhibitors, but treatment resistance occurs if the cells have a K-Ras mutation because K-Ras is downstream of EGFR in the signaling pathway.

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

C. PCR and other methods of DNA amplification have a great potential for error caused by contamination of sample or reagents with template DNA. This can derive from other samples, positive controls, or amplicons from preceding samples, but the most common source of contamination is by amplicons. Each run must contain a negative (as well as positive) control. The negative control contains all PCR reagents except the template DNA and should produce no detectable amplicons. However, PCR reactions that detect product by enzymatic, fluorescent, or chemiluminescent methods instead of gel or capillary electrophoresis will generate a signal for the negative control. This signal should be comparable to that for a substrate blank. Signals above a predetermined cutoff point will invalidate the test.

Which of the following genetic diseases is caused by an expanded trinucleotide repeat? A. Prader-Willi syndrome B. Angelman's syndrome C. Fragile X syndrome D. Williams' syndrome

C. Prader-Willi and Angelman's syndromes are most often caused by microdeletion, and Williams' syndrome is caused by a microdeletion in the gene coding for elastin. Fragile X syndrome, Huntington's disease, and myotonic dystrophy are examples of diseases caused by an expansion of trinucleotide repeats. Fragile X is so named because when cells from an affected individual are cultured in folate-deficient medium, the long arm of the X chromosome appears to have a break caused by deficient staining. The Xq27 region contains a CGG tandem sequence that can repeat up to 50 times in normal individuals. In fragile X syndrome, the repeat is extended and its length determines whether the affected persons will show mental retardation. Repeats of 50 to 230 times are associated with a carrier (premutation) state. During meiosis in females, the CGG repeat can undergo further expansion. The probability of this expansion increases with each generation. As the size of the repeat increases, so does the chance that it will cause methylation of the promoter for the FMR1 gene. The gene is needed for normal brain function and its underexpression results in mental retardation. Females in whom the premutation expands in size to a full mutation transmit the syndrome to all of their male and half of their female offspring.

An assay based on the principle of proteomics may be used for which of the following? A. Screening for colorectal cancer B. Screening for lung cancer C. Identifying malignant ovarian masses D. Identifying malignant breast tumors

C. Proteomics is the study of the proteome. Analogous to the genome, the proteome is the totality of proteins present within a cell or organism. Proteomic studies are aimed mainly at identifying protein signatures for various cancers. Serum is analyzed by time-of-flight mass spectroscopy and thousands of proteins are matched to identify those that can discriminate between cancerous and normal cells. A commercially available test based on proteomics is available for differentiating malignant from benign ovarian tumors. The test detects the presence of five proteins in serum linked to ovarian cancer, and uses multivariate statistical analysis to derive a number from 1-10 indicating the risk of cancer. It has a high sensitivity and negative predictive value.

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

C. Reverse transcriptase PCR (RT-PCR) is used to detect RNA viruses and to amplify RNA transcription products by converting the template to DNA. The master mix contains the same components needed for PCR with the addition of a heat-stable reverse transcriptase (enzyme that transcribes RNA to DNA, such as rTth DNA polymerase), manganese (a cofactor for this enzyme), and an mRNA primer. In addition to testing for infectious diseases (HIV, hepatitis C, and hepatitis E), RT-PCR is used to identify translocations in leukemia where the crossover regions are too large for efficient PCR.

Which statement about CF is accurate? A. A sweat chloride test is abnormal in all forms of CF B. Immunoreactive trypsin is deficient in all persons with CF C. Some CF mutations can cause male infertility with no other symptoms D. The CF genotype always predicts the severity of the disease

C. Serum immunoreactive trypsin is the recommended screening test for CF, but pancreatic insufficiency is not found in about 15% of CF cases. An abnormal result is confirmed by sweat chloride testing. Some infants may be too young for accurate sweat testing, and some mild forms of CF may give indeterminate results. DNA testing can be used in these cases. The CF genotype is not predictive of phenotype in most cases (an exception being ∆F508, which is almost always associated with pancreatic disease). CF mutations are responsible for about 75% of congenital bilateral absence of the vas deferens. Affected persons have at least one abnormal CF gene. Other than infertility, they are asymptomatic and may or may not have a sweat chloride level above 65 mmol/L.

Approximately how may mutations have been identified in the gene coding for the cystic fibrosis trans membrane conductor regulator protein (CFTR)? A. 10 B. 100 C. 1,000 D. 10,000

C. The CFTR protein regulates the movement of chloride across the cell membrane, and a defect in this protein results in cystic fibrosis (CF). The CFTR gene is located on the long arm of chromosome 7 and consists of 27 exons spread over 230,000 bases. The most common mutation is a deletion of three base pairs that code for phenylalanine at position 508 of the protein, ∆F508. This mutation accounts for 70% of CF genes in Whites. It causes a severe form of CF involving pancreatic insufficiency. No single test can detect all possible CF carriers and a core panel consisting of 25 probes is recommended for initial screening. The core panel is used to screen for carriers of the CF gene and can detect more than 85% of CF mutations. Since two mutations are required to produce CF, the core panel can detect approximately 80% of CF.

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

C. The PCR process results in identical copies of a piece of double-stranded DNA. The process involves three steps that are repeated to double the number of copies produced with each cycle. The first step is denaturation to separate the complementary strands. Annealing occurs when a primer binds upstream to the segment of interest on each strand, called the template. Extension involves the enzymatic addition of nucleotides to the primer to complete the new strand

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

C. The difference between a micro- and a macroarray assay is that the amount of DNA "printed" onto the substrate is larger in a macroarray assay, necessitating a larger spot. A microarray uses less than 200 μL of DNA and allows a larger number of targets to be applied. Commercially available microarrays are available that contain over 250,000 oligonucleotide spots. Short oligonucleotide targets can be synthesized on the substrate or applied by photolithography, inkjet spraying, or manually with print plates and tips that can be purchased.

A PCR analysis of a vaginal sample for Chlamydia trachomatis gives a negative result (optical density of biotinylated reaction product below the cutoff point). e 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

C. The internal control in PCR is an oligonucleotide sequence different from that of the target but that binds the same primers. Its product is detected using a different probe than is used for the target sequence. If the internal control is not amplified, this indicates an invalid test. Causes include the presence of a PCR inhibitor, denaturation of the polymerase, hybridization failure, or error in the detection system (e.g., improper pH preventing enzyme-conjugated streptavidin from acting on the substrate). The assay of this sample must be repeated.

A tissue sample for DNA analysis by PCR was processed for DNA by simple lysis and proteinase K digestion. The lysate was diluted 1:100 and its absorbance measured in an ultraviolet spectrophotometer at 260 nm and 280 nm. The absorbance ratio 260:280 was 1.2. What does this indicate? A. The DNA concentration is too high for PCR B. The DNA concentration is too low for PCR C. The sample contains too much protein D. The sample is optimal for PCR

C. The mass and purity of DNA affect the efficiency of DNA amplification. A common way to determine the amount of DNA recovered from a sample is to measure its absorbance at 260 nm, where the nitrogenous bases have an absorbance maximum. Based on the molar absorptivity of DNA at 260 nm, an absorbance of 1.0 equates to 50 ng/μL; thus, multiplying the absorbance by 50 and the dilution factor estimates the DNA concentration in ng/μL. However, proteins also absorb at 260 nm, and if present in sufficient amounts will cause overestimation of DNA and inhibit amplification. An absorbance maximum for protein is 280 nm owing to the phenolic rings of tyrosine and tryptophan. The absorbance ratio of 260:280 is a measure of protein contamination. If the ratio is below 1.7, then too much protein is present, and further purification is needed.

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

C. The melting temperature of DNA refers to the temperature required to separate the molecule into single strands. The Tm is the temperature required to convert half of the DNA from dsDNA to ssDNA. This is done by breaking the hydrogen bonds between base pairs. A-T base pairs have two hydrogen bonds, while C-G base pairs have three. Therefore, molecules with a high proportion of C-G base pairs are more resistant to heat denaturation or melting.

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

D. FISH can be used with almost any type of cell preparation, including frozen sections, formalin fixed tissues, embedded tissues, and cell suspensions such as those derived from amniotic fluid or chorionic villus sampling provided they are pure. Cells in suspension can be dropped onto glass slides or concentrated using a cytocentrifuge before processing. However, a mixture of cells from different individuals is inappropriate because the probe cannot distinguish between sources such as fetal and maternal cells.

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

D. FISH can detect conditions that are associated with structural chromosomal abnormalities and an abnormal number of chromosomes (aneuploidy). A screening test for aneuploidy employs probes labeled with different fluorescent dyes that simultaneously detect trisomy 21, 18, and 13 and the X and Y chromosomes. Deletions cause the absence of a fluorescent signal when expected, and microdeletions such as those that occur on the short arm of chromosome 5 in cri du chat syndrome can be detected by FISH. Translocations cause two different FISH probes to bind to the same chromosome. Such probes are used to identify IgH gene translocations such as t(11:14) in multiple myeloma that are of prognostic value. However, trinucleotide repeats, repetitive sequences of the same three base pairs, are not detected by FISH. This is associated with fragile X syndrome, myotonic dystrophy, Huntington's disease, and other genetic diseases. PCR or Southern blotting are used for detection of these repeats, depending upon their number.

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

D. In branched DNA (bDNA) signal amplification, the target DNA is denatured and added to a well containing immobilized probes. One end of each probe hybridizes with the target DNA, capturing it, and the other contains multiple branches that hybridize with alkaline phosphatase-labeled probes. After washing to remove the unbound labeled probes, dioxetane is added, and chemiluminescence is measured. A thermocycler is not used and the target DNA is not amplified. PCR is a licensed technology, and other methods of nucleic acid amplification have since been developed including nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), hybrid capture, and rolling circle amplification (RCA).

Which of the following alleles has the highest frequency in the general population? A. ∆F508 (cystic fibrosis) B. Factor V-Leiden (hereditary thrombophilia) C. Prothrombin G20210A (hereditary thrombophilia) D. Methylene tetrahydrofolate reductase mutation C677T homocysteinemia

D. Methylene tetrahydrofolate reductase (MTHFR) mutation is a point mutation in which thymidine replaces cytosine at nucleotide 677 in the gene. This results in a codon that substitutes valine for alanine and results in an enzyme that is more heat sensitive. The enzyme converts 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate (folate). The methyl group from the latter is transferred to homocysteine, forming methionine. In homozygotes (TT) with less than optimal dietary folate intake, deficiency of the enzyme reduces the availability of 5-methyltetrahydrofolate, causing the serum homocysteine to be increased. Such persons have an approximately threefold increased risk of coronary artery disease. In the general population, the C677T allele of MTHFR has a frequency of 30%. All of the alleles listed are of sufficiently high frequency to warrant screening of at-risk populations. The prothrombin G20210A allele has a frequency of approximately 2%, factor V-Leiden 5%, and ∆F508 approximately 3% (in Whites). Both factor V-Leiden and the prothrombin G20210A mutation result in proteins that increase the risk of thrombosis. The point mutation in factor V-Leiden results in a protein that is resistant to inactivation by protein C. The base substitution in G20210A (guanine to adenine at position 20210) results in increased transcription of the gene and overproduction of prothrombin.

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

D. Mosaicism occurs when cells within the same individual contain different numbers of chromosomes and results from nondisjunction during early embryonic development. Chimeras are molecules created when translocation occurs between genes (exons) on different chromosomes. Amplicons are exact copies of a DNA template produced by DNA amplification techniques such as the polymerase chain reaction (PCR). When a restriction enzyme cuts two different DNA molecules, the size of some fragments will differ because the number and position of restriction sites differ. Such fragments are called RFLPs for restriction fragment length polymorphisms (RFLPs). Analysis of RFLPs can be used to test for disease genes, study genetic linkage, and establish identity. It is used usually when PCR is impractical, such as when contamination occurs repeatedly or when the genes to be analyzed comprise a length of DNA too long for efficient amplification.

Which is the most sensitive method of minimal residual disease testing in chronic myelogenous leukemia? A. Karyotyping analysis B. FISH C. Flow cytometry D. RT-PCR

D. RT-PCR measures the mRNA transcript of BCR/ABL using primers to the p210 and p230 transcripts. The procedure can be done using real-time PCR with a sensitivity of 1:100,000 cells far more sensitive than karyotyping and FISH that have sensitivities of around 1:100 and 1:1,000 cells, respectively. Flow cytometry can detect 1 malignant cell per 10,000 nonmalignant cells, but a panel of antibodies is required that can differentiate malignant from normal cells. RT-PCR can also be used to evaluate the response to treatment. A 3-log decrease in copy number indicates effective treatment.

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

D. Restriction endonucleases are enzymes that cut double-stranded DNA into fragments and are important tools used in molecular diagnostics. Each restriction enzyme recognizes a specific oligonucleotide sequence, and the size and number of fragments it produces when DNA is digested depend upon the number of times that sequence is repeated in the DNA molecule. Restriction endonucleases recognize palindromic sequences (i.e., the base sequence of complementary strands reads the same from opposite directions). The sequence A-A-G-C-T-T T-T-C-G-A-A is the recognition site for HindIII, a restriction endonuclease isolated from Haemophilus influenzae. If a disease gene produces a base pair substitution at the restriction site, the enzyme will not recognize it and not cut the DNA. This results in a longer fragment that can be recognized by electrophoresis. This process was initially used to identify the hemoglobin S gene using the restriction enzyme MstII. The point mutation changes an A to a T within the restriction site, causing loss of the normal-sized fragment.

Highest-resolution HLA typing is needed for which of the following transplants? A. Heart B. Liver C. Kidney D. Bone marrow

D. Solid organ transplants require medium resolution of alleles belonging to HLA class I and class II genes. Bone marrow transplants require high-resolution typing. This involves identifying which allelic groups are present by medium-resolution testing, then sequencing of the PCR products to determine the exact alleles present.

Which statement accurately describes the clinical utility of translocation testing in leukemia? A. Relapse is predicted by any new translocation occurring after treatment B. Specific translocations associated with a type of leukemia will occur in all cases C. Translocation products for each leukemia subtype are always the same D. Translocation is a sensitive way to identify surviving leukemic cells following treatment

D. Some translocations occurring after treatment are predictive of relapse. For example, a second translocation in a person with Philadelphia chromosome-positive CML occurs in the majority of persons preceding blast crisis. However, other translocations, such as the 15:22 translocation associated with M3 AML are seen during remission and are not associated with relapse. Some translocations occur with 100% or near 100% frequency, such as 9:22 in CML and 15:17 in M3 AML. However, others occur only in some affected persons. Translocations associated with a type of leukemia are not identical in all cases. For example, the 9:22 translocation associated with CML can give rise to transcripts of different length. RT-PCR can detect as few as 1 per 105 cells containing the translocation, making translocations useful markers for detecting cells that have escaped destruction following treatment.

Which method is used to determine if the hemoglobin C gene is present in fetal cells? A. Chromosome painting B. FISH C. Restriction enzyme analysis D. PCR followed by blotting with a specific oligonucleotide probe

D. The base substitution of hemoglobin C does not affect the MstII restriction site and is not visible by FISH or other tests that detect damage to larger areas of the chromosome. PCR is used to amplify the gene region involved, and the product is tested by Southern blotting using a label-specific oligonucleotide probe.

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

D. The first three methods are probe-based PCR, while SYBR green is not. SYBR green is an intercalating dye that fluoresces when bound to dsDNA. Therefore, it can be used to quantify any PCR product, but will also fluoresce with primer dimers that may form in the PCR master mix. This can cause results to be falsely elevated. FRET (Förster or fluorescence resonance energy transfer) probes work by transfer of energy from one molecule to another. One example of FRET uses a fluorescent molecule and a quencher molecule, which—when not bound to the template—interact, resulting in no fluorescence. Binding of the primer to the target causes separation of the two molecules, resulting in excitation of the fluorescent dye by the light source.

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%

D. The human genome contains approximately 3 billion base pairs and approximately 25,000 genes. Post-transcription modification of mRNA enables production of about 100,000 proteins. However, approximately 99.9% of the DNA is homologous. The remaining 0.1% is variable and accounts for individual differences. A polymorphism is an individual difference in DNA sequence or length that occurs in at least 1% of the population. Polymorphisms arise from mutation and are transmitted to offspring. They are subject to selection pressures that cause genes to drift in the population. Over 350,000 such differences are present in the human genome, but very few are associated with human disease.

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

D. The laboratory area where manual DNA amplification methods are performed should be organized so that work flow moves from preamplification to amplification and detection. In addition to standard precautions, cotton-plugged tips are used to prevent aerosol contamination of samples. As few as 10 copies of the template introduced by accident are likely to cause a false-positive reaction. Ultraviolet light causes cross-linking of thymine bases in dsDNA, which prevents replication. This has been used as a post-PCR method of reducing contamination.

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

D. When ethidium bromide inserts between the base pairs of double-stranded DNA, the dye becomes fluorescent, releasing 480 nm light when stimulated by long wavelength ultraviolet light. Ethidium bromide staining has a sensitivity of approximately 10 ng/mL (1.5 ng per band) DNA. It is frequently added to molten agarose or capillary electrophoresis buffer at a concentration of 0.5 μg/mL in order to visualize and quantify DNA. Its binding to single-stranded DNA and RNA is not as efficient as that of more sensitive dyes such as SYBR gold, picoGreen, and YOYO-1.