Molecular Methods
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. Harr, Robert R. Medical Laboratory Science Review (Page 509). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 502). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 500). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 503). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 507). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 500). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 504). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 507). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 508). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 500). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 502). F.A. Davis Company. Kindle Edition.
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 (-ΔF/ΔT) 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. Harr, Robert R. Medical Laboratory Science Review (Page 506). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 502). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 498). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 501). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 501). F.A. Davis Company. Kindle Edition.
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). Harr, Robert R. Medical Laboratory Science Review (Page 502). F.A. Davis Company. Kindle Edition.
Which formula predicts the number of PCR products that can be produced? A. 2 n where n is the number of cycles B. N4 where N is the number of cycles C. p2 + 2pq + q 2 = 1 where p and q are the number of primers D. N2/2 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 p2 + 2pq + q2 = 1 describes the distribution of a two-allele gene in a population. Harr, Robert R. Medical Laboratory Science Review (Page 503). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 505). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 499). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 505). F.A. Davis Company. Kindle Edition.
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). Harr, Robert R. Medical Laboratory Science Review (Page 507). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 498). F.A. Davis Company. Kindle Edition.
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 fluorescentlabeled 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. Harr, Robert R. Medical Laboratory Science Review (Page 503). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 502). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 509). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 506). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 509). F.A. Davis Company. Kindle Edition.
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 T m 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. Harr, Robert R. Medical Laboratory Science Review (Page 497). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 508). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 508). F.A. Davis Company. Kindle Edition.
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). Harr, Robert R. Medical Laboratory Science Review (Page 503). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 498). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 497). F.A. Davis Company. Kindle Edition.
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. Harr, Robert R. Medical Laboratory Science Review (Page 499). F.A. Davis Company. Kindle Edition.
