SUCCESS! in Clinical Laboratory Science - Molecular Diagnostics (26-50)

The coding region of a human gene is called A. Exon B. Intron C. SNP D. VNTR

A. Exon A. The coding regions of eucaryote genes are called exons. The noncoding intervening regions are called introns. In eucaryotes, the introns and exons are transcribed into mRNA; however, before mRNA is translated, the introns are removed and the exons are spliced together. SNP is an abbreviation for single nucleotide polymorphism, and VNTR refers to variable number tandem repeats.

When comparing two dsDNA sequences of equal length, the strand that has a higher A. G + C content has a higher melting temperature (Tm) B. A + T content has a higher Tm C. A + T content has more purines than pyrimidines along its length D. G + C content has more purines than pyrimidines along its length

A. G + C content has a higher melting temperature (Tm) A. DNA is composed of two strands of polynucleotides coiled in a double helix. The outside backbone is composed of sugar-phosphate moieties, whereas the purine and pyrimidine bases are stacked inside the helix. The size and stability of the DNA molecule is such that only specific bases can hydrogen bond to each other to hold the two strands together (A-T, C-G, and vice versa). This is referred to as complementary base pairing. An A-T base pair is less stable than a C-G base pair, because three hydrogen bonds form between C-G and only two hydrogen bonds form between A-T. The increased stability between C-G causes the melting temperature (Tm) to be greater in a double-stranded DNA (ds DNA) segment with more C-G pairs than a segment with more A-T pairs. In all dsDNA molecules, the number of purines (A + G) equals the number of pyrimidines (C + T).

For the purpose of diagnosing genetic diseases, what component of whole blood is used for the extraction of DNA? A. Leukocytes B. Plasma C. Platelets D. Red blood cells

A. Leukocytes A. Leukocytes are routinely used for extraction of DNA from human blood. Mature red blood cells and platelets have no nuclei. Plasma or serum can be used for detection of viremia, but it is not used for analysis of genetic diseases.

The assay method that detects the expression of a gene rather than the mere presence or structure of a gene is termed A. RT-PCR B. TMA C. Multiplex PCR D. Ribotyping

A. RT-PCR A. Reverse transcription-polymerase chain reaction (RT-PCR) is used to detect gene expression; genes are expressed by transcription into mRNA. The starting material for RT-PCR is mRNA. The only method listed whose target sequence is found in mRNA is RT-PCR. Transcription mediated amplification targets are usually ribosomal RNA. In ribotyping, rRNA probes detect ribosomal RNA genes present in total bacterial DNA; bacteria can be grouped on the basis of banding patterns that result. Multiplex PCR describes a method in which DNA is the target or template, and several different primer sets are included in the reaction mix. An example of multiplex PCR would include methods that detect Chlamydia trachomatis and Neisseria gonorrhoeae in one reaction mix

One method to prevent "false-positive" PCR results includes the use of dUTP in the reaction mix, resulting in amplicons containing U in place of T. The enzyme used to decrease contamination is A. Uracil-/V-glycosylase B. Tag polymerase C. SI nuclease D. DNase

A. Uracil-/V-glycosylase A. The sensitivity of amplification techniques can be viewed as a double-edged sword. On one hand, the techniques have allowed detection of genetic sequences that are found in limited numbers within a sample. However, because the method creates large amounts of target sequence, the areas within the laboratory can become contaminated with amplicons. Amplicon contamination produces false positive results. The use of dUTP in the reaction mix results in PCR products (i.e., amplicons) containing uracil in place of thymidine. The enzyme used to decrease contamination of previously generated dU-containing amplicons is uracil-/V-glycosylase (UNG). Samples are pretreated with this enzyme before their use in subsequent PCR reactions to remove contaminating dU-containing amplicons if present. Pretreatment with UNG has no effect on sample DNA containing thymidine residues. Other procedures necessary to avoid contamination include dedicated areas for reagent preparation, impeccable technique, amplification and post-amplification analysis, and use of aerosolbarrier pipette tips. Treatment of work surfaces, equipment, and pipettors with UV light can also be used to prevent contamination.

For questions 45-47, refer to Color Plates 58a and b What temperature range is most appropriate for Step 2? A. 25-35°C B. 55-65°C C. 70-80°C C. 90-100°C

B. 55-65°C

The component parts of a dNTP include a purine or pyrimidine base, a A. Ribose sugar, and one phosphate group B. Deoxyribose sugar, and three phosphate groups C. Ribose sugar, and two phosphate groups D. Deoxyribose sugar, and two phosphate groups

B. Deoxyribose sugar, and three phosphate groups B. dNTP stands for deoxyribonucleotide triphosphate. Nucleotides are the building blocks of nucleic acids. They are composed of phosphate groups, a 5-sided sugar molecule, and a nitrogenous base. Nitrogenous bases are either purines (A, G) or pyrimidines (C, T, or U, an RNA-specific base). The sugar molecules are either ribose (in RNA) or deoxyribose (in DNA), with the only difference in structure being the lack of a hydroxyl group at position 2' in the deoxyribose molecule. When the sugar is bound to a base without the phosphate group, the molecule is called a nucleoside. A nucleotide can have 1, 2, or 3 phosphate groups, which are termed monophosphate, diphosphate, and triphosphate, respectively.

An ordered sequence of events makes up the cell cycle. Which of the following describes the correct sequence of events starting at Gl? A. G1,G2, S,M B. G1,S,G2,M C. G1,M,G2, S D. G1,S,M, G2

B. G1,S,G2,M B. Most of the lifetime of a cell is spent in Gl phase, during which the cells can produce their specialized proteins and accomplish their essential functions. However, when the signal is received for cell division, the cell enters S phase. In S phase the DNA in all chromosomes is duplicated. At the end of S phase, the duplicated chromosomes remain attached at the centromere. A time delay, G2, separates events of the actual separation of individual chromosomes from their duplicated pairs. Next, the M phase or mitosis is when the two members of each pair of chromosomes go to opposite ends of the original cell. This separates 46 chromosomes into two sets of 23 in each cell. Finally, a cleavage furrow is formed and separates the original cell into two daughter cells. Each cell contains a copy of all the genetic information from each parent

Frequently, DNA probes are used to detect target sequences in Northern or Southern blots. Hybridization occurs between DNA probe and RNA or DNA on the blot, respectively. To ensure that only exactly matched complementary sequences have bound together, the blot is washed under stringent conditions. Stringency of the wash steps to remove unbound and mismatched probe can be increased by A. High temperature, high NaCl concentration, and high detergent (i.e., SDS) solution B. High temperature, low NaCl concentration, and high detergent (i.e., SDS) solution C. High temperature, high NaCl concentration, and low detergent (i.e., SDS) solution D. Low temperature, high NaCl concentration, and high detergent (i.e., SDS) solution

B. High temperature, low NaCl concentration, and high detergent (i.e., SDS) solution B. Stringency of hybridization is accomplished at two steps in the blotting technique. The first step is hybridization conditions of the labeled probe in solution with the transferred RNA or DNA targets on the membrane. The second step occurs when the membrane is washed to remove unbound probe. In the hybridization reaction, formamide and temperature can be used to increase stringency. During wash steps, increasing temperature and increasing detergent concentration (e.g., 1% SDS) will increase stringency; whereas lowering NaCl concentration also increases stringency. At the end of the highest stringency wash, only specific hybrids of interest should remain on the blot.

Which of the following statements best describes characteristics of RNase? A. It degrades mRNA but not rRNA. B. It is found in large concentrations on hands. C. Its activity can be eliminated by autoclaving. D. Its activity occurs in a limited temperature range between 25 and 65°C.

B. It is found in large concentrations on hands. B. The highest concentration of RNase is found on hands; thus, it is imperative that gloves be worn when working with RNA. RNases are ubiquitous and can act at temperatures below freezing (-20°C) and above boiling. For longterm storage, purified RNA is best stored at —70°C or below. RNases plague experiments in which RNA is used. Simple autoclaving does not eliminate RNase activity. To remove RNases, glassware must be pretreated with an RNase inhibitor, such as DEPC, followed by autoclaving; alternatively, baking glassware in a >250°C oven for 4 hours will destroy RNase. To prevent RNA degradation, isolation of RNA should be done using chaotropic agents (e.g., guanidine isothiocyanate) that inhibit RNase activity. When analyzing RNA in a gel, formaldehyde or other agents that denature RNases must be included in the gel. High-quality (i.e., undegraded) RNA will appear as a long smear with two or three distinct areas that correspond to the ribosomal RNA subunits: 28S (-4800 bases), 18S (-1800 bases), and 5.8S (-160 bases), whereas degraded RNA will appear as a smear at the bottom of the gel

Which of the following amplification methods does not employ isothermal conditions? A. Nucleic acid sequence-based amplification (NASBA) B. Polymerase chain reaction (PCR) C. Strand displacement amplification (SDA) D. Transcription mediated amplification (TMA)

B. Polymerase chain reaction (PCR) B. PCR requires a thermocycler because cycling at three different temperatures is the basis for this technique. First, template DNA (i.e., which may contain the target sequence) is denatured at 94°C. Next, the temperature is lowered to allow specific primers to anneal to the single-stranded target, generally at temperatures near 55 °C. In the third portion of the cycle, primers are extended using deoxynucleotide triphosphate molecules to form a complementary copy of DNA under the direction of a thermostable DNA polymerase enzyme, such as Tag polymerase. The optimal temperature at which Tag polymerase acts to extend the primers is 72°C. Thus, at the end of one cycle, one molecule of dsDNA has now become two molecules of dsDNA. Cycles are generally repeated about 30 times to theoretically yield 230 DNA molecules. The three steps of each cycle are termed denaturation (94°C), annealing of primers (~55°C), andextension of primers (72°C). The other methods listed, nucleic acid sequence-based amplification, strand displacement amplification, and transcription mediated amplification, are also amplification methods; however, they have been modified so all reactions take place at a single temperature (isothermal).

Which sample in Color Plate 60• contains the largest amount of cytomegalovirus? A. Sample 4 B. Sample 5 C. Sample 11 D. Only qualitative results can be determined in this assay.

B. Sample 5 B. Color Plate 60 • is graphic display of a realtime PCR (i.e., qPCR) run for cytomegalovirus (CMV). Real-time PCR assays can measure the amount of starting target sequence (i.e., template in sample) accurately. Rather than measuring PCR product generated at the stationary or endpoint of the PCR assay, qPCR analysis is done as PCR products are formed (i.e., during the exponential phase) where accumulation of fluorescence is inversely proportional to the amount of starting template (i.e., the shorter the time to accumulate signal, the more starting material). Optimal threshold level is based on the background or baseline fluorescence and the peak fluorescence in the reaction and is automatically determined by the instrument. Using 10-fold dilutions of known positive standards, a standard curve can be made. The qPCR cycle at which sample fluorescence crosses the threshold is the threshold cycle (CT). Using the standard curve, the starting amount of target sequence in each sample can be determined by its CT. Fluorescence versus CT is an inverse relationship. The more starting material, the fewer cycles it takes to reach the fluorescence threshold (i.e., large amounts of fluorescence accumulate in a short time). The CT for sample 5 is 21, sample 4 is 25, and sample 11 is 38; therefore, sample 5 has more CMV copies than sample 4, which has more CMV copies than all the other samples with CT values indicated, including sample 11 with the least CMV. The samples below the threshold fluorescence of 30 are negative for CMV.

An advantage of amplification technologies for clinical laboratories is that A. They require inexpensive test reagents B. They lend themselves to automated methods C. Each target molecule sought requires a unique set of primers D. Contamination is not a concern when performing these assays

B. They lend themselves to automated methods B. Amplification methods can be automated and standardized, which is proven by the variety of test systems presently on the market. Amplification methods are very sensitive and theoretically can detect one target DNA molecule in a sample. However, increased sensitivity raises the likelihood of false positive results due to contamination of testing areas with PCR amplicons. In addition, most amplification methods can be completed within 4-6 hours and can detect microorganisms that do not grow readily by standard culture techniques. At this time, test reagents are still quite expensive, although if decreased turn-around time would translate into shorter hospital stays, then resultant healthcare costs could be reduced by use of these methods in the clinical laboratory. A disadvantage of amplification technologies is that they require a unique set of primers for each target DNA being sought. Thus, amplification techniques may be replaced by use of DNA microarrays because thousands of genes can be assessed at one time, rather than a limited number of molecules of interest being assayed

For questions 45-47, refer to Color Plates 58a and b What substance within the PCR mix influences the accuracy of cDNA? A. Oligonucleotide primers B. Monovalent cation K+ C. Divalent cation Mg2+ D. Deoxyribonucleotide triphosphate molecules

C. Divalent cation Mg2+

In Color Plate 57 the procedure of Southern blotting is diagrammed. In the upper panel, restricted genomic DNA fragments have been separated by electrophoresis in an agarose gel. In lane 1 is a molecular weight marker, in lanes 2-A are three patient samples, and in lane 5 is a positive control DNA sequence for the probe used. After electrophoresis, DNA was transferred from the gel onto a nylon membrane and then hybridized with a radiolabeled probe that recognizes CGG trinucleotide repeat. Fragile X syndrome is the most frequently inherited form of mental retardation in males (1:1000-1:1500 individuals). In affected individuals, expansions of the trinucleotide repeat within the fragile X gene increase to greater than 200 repeats. The bottom panel shows the resultant autoradiogram after a series of high-stringency washes. The three patient samples (lanes 2-4) are DNA from individuals of a single family, one of them suffering from fragile X syndrome. In which lane is the mentally handicapped patient's sample? A. Lane 2 B. Lane 3 C. Lane 4 D. Cannot be determined by the results given

C. Lane 4 C. Refer to Color Plate 57. Given that the probe used will recognize the trinucleotide repeat found in the fragile X gene, FMR-1, the location of positive signals will give information about the size of the repeat sequence within each person's DNA. The normal allele for FMR-1 has 6 50 trinucleotide repeats (found in normal individuals), the premutation for FMR-1 contains 50-200 trinucleotide repeats (found in unaffected individuals), and the disease allele (found in affected individuals) has >200 repeats. Because electrophoresis separates DNA by size such that the larger fragments travel shorter distances than smaller fragments, then the larger fragment in the affected individual caused by the expansion of the trinucleotide repeat would be represented in Color Plate 57 by lane 4 of the diagram.

The following question refers to Color Plate 59B. Factor V Leiden mutation causes increased risk of thrombosis. It is caused by a single base mutation in which guanine (G) is substituted for adenine (A) with a subsequent loss of a restriction site for the enzyme MnH. Primers used in this example generate a 223 bp PCR product from patient DNA. After resulting PCR products are digested with Mn/I, normal patients produce the following DNA fragments: 104 bp, 82 bp, 37 bp. In Color Plate 59m, the 37 bp fragment is not seen in all lanes because it is sometimes below detectable levels. Lane identities are as follows: M (molecular weight marker), 1-5 (patient 1 to patient 5, respectively), + (positive control showing 104, 82, and 37 bp fragments), Neg (sterile water used in place of sample DNA). Which patient is heterozygous for the factor V Leiden mutation? A. Patient 1 B. Patient 2 C. Patient 3 D. Patient 4

C. Patient3 C. Refer to Color Plate 59m. Factor V Leiden mutation (A506G) causes activated protein C resistance that results in increased risk of hypercoagulability. The mutation destroys a Mn/I restriction enzyme site in an amplified 223 bp PCR product from patient DNA. From the electrophoretic pattern, wild-type or normal factor V will show three bands after Mn/I digestion (104 bp, 82 bp, 37 bp), as in patients 1, 4, and 5. The pattern seen with patient 2 is that of a homozygous mutant with two bands (141 bp and 82 bp). In the heterozygous patient 3, one allele is normal and the other is mutant. Thus, the banding pattern results in four bands (141 bp, 104 bp, 82 bp, and 37 bp). Sometimes the 37 bp fragment band is not seen because it is below detectable levels

Molecular typing of bacterial strains is based on restriction fragment length polymorphisms (RFLPs) produced by digesting bacterial chromosomal DNA with restriction endonucleases. Which of the following techniques is used to separate the large DNA fragments generated? A. Ribotyping B. DNA sequencing C. Pulsed field gel electrophoresis D. Reverse transcription-polymerase chain reaction

C. Pulsed field gel electrophoresis C. Pulsed field gel electrophoresis (PFGE) is used to separate extremely large DNA molecules by placing them in an electric field that is charged periodically in alternating directions, forcing the molecule to reorient before moving through the gel. Larger molecules take more time to reorient; thus they move more slowly. Bacterial DNA is digested by restriction enzymes in agarose plugs. The PFGE of the digested fragments provides a distinctive pattern of 5 to 20 bands ranging from 10 to 800 kilobases. DNA sequencing determines the exact nucleotide sequence base by base of any organism; however, it is too laborious for epidemiologic purposes. Ribotyping is a Southern blot type of analysis using rRNA probes to detect ribosomal operons (i.e., sequences coding for 16S rRNA, 25S rRNA, and one or more tRNAs) of individual bacterial species. Its discriminatory power is less than PFGE. Reverse transcription-polymerase chain reaction (RTPCR) is a method that determines whether a gene is being expressed. The starting material for RTPCR is ssRNA.

Purified DNA remains stable indefinitely when stored as A. Small aliquots at 4°C B. Large aliquots at 25°C C. Small aliquots at -70°C D. Large aliquots at -20°C

C. Small aliquots at -70°C C. Purified DNA is relatively stable provided it is reconstituted in buffer that does not contain DNases. Therefore, high-quality reagents and type I sterile water should be used in preparing buffers used for this purpose. Experiments have shown that purified DNA is stable for as long as 3 years at refrigerated temperature (4°C). However, long-term storage of purified DNA is best accomplished at -20 to -70°C in a freezer that is not frost free to avoid freeze-thaw cycles that may damage DNA and by dividing the original DNA sample into multiple small aliquots for storage.

The central dogma is that DNA is used to make RNA, which is then used to make protein. In this scheme the two processes that are involved (i.e., DNA to RNA and RNA to protein) are termed A. Replication and transcription B. Synthesis and encryption C. Transcription and translation D. Initiation and elongation

C. Transcription and translation C. Central dogma describes the flow of genetic information from DNA to RNA to protein. Individual DNA molecules serve as templates for either complementary DNA strands during replication or complementary RNA molecules during transcription. In turn, RNA molecules serve as templates for ordering of amino acids by ribosomes to form polypeptides during protein synthesis, also known as translation.

How many chromosomes are contained in a normal human somatic cell? A. 22 B. 23 C. 44 D. 46

D. 46 D. DNA in human somatic cells is compartmentalized into 22 pairs of chromosomes, referred to as autosomes. They are numbered 1 through 22. In addition, humans have two sex chromosomes, both an X and Y (in males) or two X chromosomes (in females). Thus, the total number of chromosomes is 46 in a normal diploid cell. The genetic information of one set of chromosomes comes from the mother of the individual and the other set from the father. Gametes (i.e., eggs and sperm) are haploid and contain only one set of chromosomes (23 chromosomes in human gametes), so that upon fertilization, a diploid zygote is formed

For questions 45-47, refer to Color Plates 58a and b What temperature is best for use in Step 1? A. 35°C B. 55°C C. 75°C D. 95°C

D. 95°C

Which of the following assays cannot be accomplished using PCR methods employing only Tag polymerase? A. Diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae infection B. Detection of single base pair gene mutations, such as in cystic fibrosis C. Detection of HLA-A, B and DR genotypes D. Determination of viral load for HCV

D. Determination of viral load for HCV D. Hepatitis C vims (HCV) has an RNA genome, and thus a reverse transcription step is needed to convert RNA into complementary DNA for use in the subsequent PCR that makes multiple copies of the target sequence. RT-PCR is both highly specific and sensitive. Viral load testing also requires that the methodology be quantitative. Quantification can be accomplished by qPCR techniques or by inclusion of a known amount of a synthetic nucleic acid, a quantification standard (QS), in the sample. The QS binds the same primers as the viral target, and so the kinetics of amplification for both may be assumed to be approximately equal. The viral target and QS are coamplified in the same reaction, and the raw data are manipulated mathematically to determine the viral load present in the specimen. To detect genetic sequences specific for the human leukocyte antigen (HLA) loci, bacteria, and gene mutations, the starting material is usually DNA; therefore, PCR methods, rather than RT-PCR, would be employed.

Which of the following is the least likely inhibitor of PCR? A. Heme B. Sodium heparin C. DEPC (diethylpyrocarbonate) D. EDTA (ethylenediaminetetraacetic acid)

D. EDTA (ethylenediaminetetraacetic acid) D. EDTA and ACD (acid citrate dextrose) are the preferred anticoagulants for specimens that will undergo PCR. These reactions can be inhibited by a variety of substances. PCR inhibitors are concentration dependent; inhibition can often be overcome by simply diluting the DNA sample. Heme and sodium heparin can inhibit PCR. However, laboratory methods can be used to remove these inhibitors, if necessary. Diethylpyrocarbonate (DEPC) is a substance used to inhibit RNases; it can also inhibit PCR.

The translocation resulting in the Philadelphia chromosome is detected by A. Southern blot analysis only B. Cytogenetic analysis (e.g., karyotyping) only C. PCR, Southern blot, and cytogenetic analysis D. RT-PCR, Southern blot, and cytogenetic analysis

D. RT-PCR, Southern blot, and cytogenetic analysis D. The translocation resulting in the Philadelphia chromosome can be detected by reverse transcription-polymerase chain reaction (RTPCR), Southern blot, and cytogenetic analysis. The presence of a Philadelphia (Ph) chromosome confirms the diagnosis of chronic myelogenous leukemia (CML). The Ph chromosome is a shortened chromosome 22 that arises from a reciprocal translocation involving the long arms of chromosomes 9 and 22. This translocation involves the proto-oncogene c-ABL, normally present on chromosome 9q34, and the BCR gene on chromosome 22ql 1. The juxtaposition of ABL with BCR results in the formation of a BCR-ABL fusion gene, which is subsequently transcribed into a chimeric BCR-ABL mRNA that is ultimately translated into a chimeric BCR-ABL protein product. Traditionally, this rearrangement can be seen cytogenetically by visualization of the patient's karyotype (i.e., metaphase spread of patient's chromosomes). Recent techniques have been developed in which fluorescent-labeled probes for this gene rearrangement can be used to probe the patient's metaphase or interphase spread, called fluorescence in situ hybridization (FISH). Molecular methods to check for this gene rearrangement include Southern blotting and RT-PCR. PCR cannot be used for this particular gene rearrangement because ECR/abl breakpoints span large segments of DNA, which prevents direct PCR testing. Instead, RT-PCR is used. The BCR/abl chimeric mRNA is used as a template because primer annealing sites in the breakpoint region of the mRNA are a smaller size, suitable for amplification.

In RNA, which nucleotide base replaces thymine of DNA? A. Adenine B. Cytosine C. Guanine D. Uracil

D. Uracil D. The four nucleotide bases found in RNA are adenine (A), guanine (G), cytosine (C), and uracil (U). The purines A and G are the same as in DNA. C is present in both DNA and RNA; however, in RNA, the DNA nucleotide base thymine (T) is replaced by uracil (U). RNA is usually single stranded, although double-stranded areas can occur. A pairs with U, and C pairs with G.