Success in CLS Ch. 10 Molecular Diagnostics

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

45. D

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 d. 90-100°C

46. 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

47. C

Which of the following is characteristic of DNA chips (i.e., DNA microarrays)? a. Allow detection and discrimination of multiple genetic sequences at the same time. b. Thousands of oligonucleotide probes are labeled and placed on glass or silicon surfaces. c. Unlabeled target sequences within the patient sample are detected by hybridization to labeled probes. d. All the above

A. DNA chips (i.e., DNA microarrays) allow detection and discrimination of multiple genetic sequences at the same time. DNA chips have thousands of oligonucleotide probes arranged on glass or silicon surfaces in an ordered manner. Target sequences within the patient sampleare fluorescently labeled in solution. The labeled sequences in solution are then incubated with the DNA chips containing the oligonucleotide probes attached to the silicon or glass surface. Hybridization will occur between labeled complementary sequences within the patient samples and their corresponding probe on the chip. The DNA chip is placed in an instrument that scans the surface with a laser beam. The intensity of the signal and its location are analyzed by computer and provide a quantitative description of the genes present. Because placement of the oligonucleotides is known, identification of the gene or organism may be determined.

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. 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 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 (dsDNA) segment with a 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).

In the isolation of RNA, diethylpyrocarbonate (DEPC) is used to a. inhibit RNase b. Lyse the cells c. Precipitate the DNA d. Remove buffer salts

A. In the isolation of RNA, it is very important to remove all RNase activity. Such enzymes are considered ubiquitous, so precautions must always be taken. Diethylpyrocarbonate (DEPC), or diethyl oxydiformate, will inactivate RNase, thus protecting RNA from degradation. Is it used in solution at 0.1-0.2% (w/v) concentration.

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 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.

"Star activity" for a restriction enzyme refers to a. An ability to cleave DNA at sequences different from their defined recognition sites b. The enzyme's specificity for site of methylation within the nucleotide sequence c. The temperature and pH conditions at which the enzyme will function optimally d. The percent increased accuracy of the enzyme when placed in ideal conditions of pH

A. Restriction enzymes will show specificity for a target nucleotide sequence when used under optimal conditions of temperature and glycerol, salt, and substrate concentrations. If these conditions are not optimal, some enzymes will lose their specificity and begin to cleave more randomly. This undesirable, nonoptimal digestion is called "star activity." Such activity is evident when the following parameters are altered in the reaction environment: more than 100 units of enzyme per microgram of DNA, more than 5% glycerol content, less than 25 mM salt concentrations, pH > 8.0, presence of dimethyl sulfoxide (DMSO), ethanol, or other organic solvents.

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. 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.

When compared to Souther blot hybridization testing, PCR a. is less sensitive to DNA degradation than Southern blot b. includes transfer of DNA onto a nylon membrane c. Requires no specialized equipment d. Is more labor intensive

A. Standard Southern blot techniques recommend the use of 10 micrograms of high-quality genomic DNA when studying single-copy genes. In a subsequent step the genomic DNA is restricted (i.e., cut into small fragments of predictable size). The resulting fragments of the gene of interest generally range in size from 1.0 to 10.0 kilobases. In contrast, the gene sequence of interest to be amplified in a routine PCR tragets a smaller portion of the gene (generally 150-500 bases in length). Because the target is a smaller size, partially degraded DNA (i.e., genomic DNA samples of lesser quality) can be amplified successfully. Long-range PCR methods are available that extend the range of PCR products synthesized from 5 to 35 kilobases. Because PCR targets are usually a few hundred bases in length, high-molecular-weight DNA is not necessary for successful PCR. It requires a thermocycler to take the reaction through the cycles of three temperatures needed for denaturation, hybridization, and elongation steps. Turn-around time is also an advantage of PCR reactions because results can be completed in less than 4 hours, whereas Southern blotting takes up to 1 week to complete because of multiple steps required for this procedure.

The coding region of a human gene is called a. exon b. intron c. SNP d. VNTR

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

In naming restriction endonucleases, the first letter of the name comes from the a. Bacterial genus b. Bacterial species c. Scientist who discovered it d. Geographic location of its discovery

A. The first letter of a restriction endonuclease's name comes from the bacterial fenys from which it originated. The second and third letters derive from the bacterial species. The last letter indicated the subspecies or strain from which the enzyme was obtained. The last Roman numeral represents the numerical place the enzyme has among those which have been isolated from that bacterial genus/species/strain. For example, EcoRI is the first restriction endonuclease isolated from the bacterium Escherichia coli, strain R, whereas EcoRV is the fifth such enzyme to be discovered.

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-N-glycosylase b. Taq polymerase c. S1 nuclease d. DNase

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-N-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, impeccable technique, amplification and post-amplification analysis, and use of aerosol-barrier pipette tips. Treatment of work surfaces, equipment, and pipettors with UV light can also be used to prevent contamination.

Which of the following is commonly used as a label in molecular tests? a. Biotin b. DNase c. RNase d. 125I

A. There are three essential parts to any molecular test performed: (1) a target, (2) a probe, and (3) a signal that can be detected. There are many ways a probe can be labeled in order for a signal to be produced and an analyte measured. Radioactive isotopes, such as 32P, 33P, 35S, and 125I, have traditionally been used to label probes. Positive signals are measured using X-ray exposure or scintillation counting. However, because of environmental factors, costs, and safety concerns, radioactive labels are being used with decreasing frequency. Nonradioactive probes are often labeled with haptens (e.g., digoxigenin), biotin, fluorescein, rhodamine, or a chemical such as acridinium esters. Detection of the hybridization (i.e., a positive test) is dependent on the type of label used, but it is generally colorimetric, fluorescent, or chemiluminescent. Hapten-labeled and biotin-labeled probes are detected by enzyme-conjugated antihapten antibodies and enzyme-conjugated streptavidin. Enzyme conjugates used are horseradish peroxidase and alkaline phosphatase.

If a DNA probe is added to nitrocellulose after the transfer step but before the blocking step, which of the following will occur? a. The probe will nonspecifically bind to its DNA target. b. Unoccupied spaces on the nitrocellulose will bind the probe. c. The DNA target on the nitrocellulose will be unable to bind the probe. d. Bound probe will be washed away in the next wash step.

B. After DNA is transferred to a nitrocellulose or nylon membrane, many sites on the membrane will not be occupied. Adding a probe at this point will not only allow for specific binding of the probe to the target DNA sequence, but also the nonspecific binding of the probe to the available binding sites on the membrane. This will cause nonspecific signal generation throughout the matrix. To prevent this, the membrane must first be treated with blocking agents. Denhardt solution and denatured nonhomologous DNA (e.g., salmon sperm DNA) are often used to bind up all the available sites on the matrix and allow for specific binding of the probe in the next step.

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. 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 on target DNA molecule in a sample. However, increases 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.

If 20% of the nucleotides in an organism are adenine, predict the percentage of nucleotides that are guanine. a. 20% b. 30% c. 40% d. 60%

B. Because of the base pairing property within DNA, the presence of 20% adenine (A) means there must also be 20% thymine (T) in the organism. This means 40% of the DNA is A or T, leaving 60% of the DNA to be cytosine (C) or guanine (G). Because there must be an equal amount of each base type within the base pair, 60% divided by 2 gives 30% each of cytosine and guanine.

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. Color plate 60 is graphic display of a real-time 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 end-point 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 ordered sequence of events makes up the cell cycle. Which of the following describes the correct sequence of events starting at G1? a. G1, G2, S, M b. G1, S, G2, M c. G1, M, G2, S d. G1, S, M, G2

B. Most of the lifetime of a cell is spent in G1 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 separate 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.

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 medicated amplification (TMA)

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 triphophate molecyles to form a complementary copy of DNA under the direction of thermostable DNA polymerase enzyme, such as Taq polymerase. The optimal temperature at which Taq 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 2^30 DNA molecules. The three steps of each cycle are termed denaturation (94°C), annealing of primers ~55°C), and extension 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).

Purification resinsused to isolate DNA take advantage of the fact that DNA is a. Double stranded b. Negatively charged c. Higher in concentration than RNA d. Higher molecular weight than RNA

B. Resin-based purification of DNA takes advantage of the fact that DNA, at alkaline pH, possesses a net negative charge. Cells are incubated with detergent, which causes their lysis. RNA is digested with RNase, and the solution is neutralized with potassium acetate. This salt solution will precipitate the detergent and all proteins. Lysate is added to the exchange resin, and both DNA and residual RNA will bind. RNA and ssDNA are removed with a wash buffer, and the desired dsDNA will be eluted from the resin with either water or pH 8.0-8.5 buffer.

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. 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 degrees C.

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 long-term 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 myst be pretreated with an RNase inhibitor, such as DEPC, followed by autoclaving; alternatively, baking glassware in a >250°C overn 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.

After performance of DNA electrophoresis, the isolated bands in the kilobase size range appear too close together. Which of the following can be done with the next run to improve the appearance/separation of the bands in the samples? a. Increase the percent agarose concentration of the matrix b. Increase the running time of the electrophoresis assay c. increase the sample volume applied to the gel d. Decrease the sample volume applied to the gel

B. The rate of electrophoretic separation when using polyacrylamide or agarose gels is affected by time, current, and the percent matrix used. Sample volume will not affect rate of separation but only makes the resulting bands more visible when stained. Achieving increased separation can be accomplished by increasing the time or current used. It can also be achieved by decreasing the percent matrix, because the "pores" present in a 1% agarose gel will be larger than those in a 5% gel. This larger size pore will allow easier molecular passage of DNA molecules during electrophoresis. Conversely, achieving a tighter band pattern (i.e., higher resolution of smaller DNA molecules) can be accomplished by decreasing time or current, or increasing percent matrix used.

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. 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 pyriminides (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.

In forensic testing, DNA fingerprinting can identify individuals with high accuracy because a. Human genes are highly conserved b. Only a small amount of sample is needed c. Human gene loci are polymorphic d. DNA is stable and not easily contaminated

C. Although human genes are highly conserved in gene coding regions, human gene loci are polymorphic, which means many forms of the gene can exist at a given locus, making each person "unique." Only identical twins are not "unique." Short tandem repeats (STRs) account for the many polymorphisms used in DNA fingerprinting. STRs are short, repetitive sequences of 3-7 base pairs and are abundant in the human genome. There are STR kits commercially available from several manufacturers. The common loci used in forensics to obtain DNA fingerprints are the CTT triplex and the FFv triplex. These contain the following loci: CSF1PO (protooncogene CSF-1), TH01 (tyrosine hydroxylase gene), F13A01 (coagulation factor XIII gene), FES/FPS (proto-oncogene c-fes/fps), and v-WA (von Willebrand gene). This testing does not require large quantities or high-quality DNA for successful results. It uses PCR, which is highly sensitive; however, this characteristic also makes the PCR method prone to contamination.

What is the theoretic estimation of the number of DNA target sequences present (per original double-stranded DNA in solution) following 15 cycles of PCR? a. 30 b. 2^10 (i.e., 1024) c. 2^15 (i.e., 32,768) d. 2^20 (i.e., 1,048,576)

C. Assuming 100% efficiency, each cycle of the polymerase chain reaction doubles the number of DNA molecules present in the solution. Starting with one DNA template molecule, there would be 2^2 = 4 DNA molecules present after two cycles. After 5 cycles, this would result in 2^5 = 32. Based on a starting single molecules of double-stranded DNA, after 15 cycles there would theoretically be 2^15 molecules (32,768). Actual yield is somewhat less than theoretical yield because PCR products created in the first two PCR cycles are slightly longer than the target amplicon. Thus, yield may be better calculated as 2^(n-2). Actual yield may be decreased by a plateau effect that may occur in later PCR cycles when some components of the PCR become reaction limiting.

The most useful feature of the molecules streptavidin and biotin is that they bind a. Specifically to nucleid acids b. Only in neutral pH conditions c. To each other with very high affinity d. Directly to DNA immobilized on nitrocellulose

C. Biotin is a vitamin involved physiologically in single carbon transfers. Streptavidin is a protein derived from Streptomyces avidinii, consisting of four subunits, each of which can bind one biotin molecule. This bond formation is rapid and essentially irreversible. The interaction between streptavidin and biotin is the strongest known noncovalent biologic interaction between a protein and its ligand. In vitro assays take advantage of this strong and specific binding by covalently attaching streptavidin to a reporter molecule (e.g., a primary antibody) and then incubating this with a secondary fluorescent-labeled antibody conjugated to biotin. Each streptavidin molecule will bind four biotin-conjugated molecules, thereby increasing four-fold the signal generated.

The central dogma is that DNA us 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. 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.

What enzyme recognizes and cuts overlapping DNA sequences formed between mutant or normal probes and target sequences within samples? a. Restriction endonuclease b. DNA ligase c. Cleavase d. RNase H

C. Cleavase is an enzyme isolated from bacteria that is likely important in DNA repair in vivo. The enzyme recognizes overlapping sequences of DNA and cleaves in the overlapping sequence. Third Wave Technologies has exploited the use of this enzyme in their Invader system. Target nucleic acid is mixed with Invader and signal probes. When the Invader and signal probes bind the target, the 5' end of the signal overlaps with the Invader probe, and cleavase cleaves the signal probe. In the next step, the cleaved signal probe binds a fluorescent-labeled reporter probe containing complementary sequences and a quencher molecule, thus forming an overlapping structure. This molecule is subsequently cut by cleavase, which removes the reporter molecule from the quencher. The signal generated is directly related to the amount of target sequences in the original sample. Restriction endonucleases are also bacterial enzymes that recognize specific sequences within DNA and cut DNA near or within the recognized sequence. DNA ligase catalyzes the formation of a phosphodiester bond between adjacent 3' hydroxyl and 5' phosphate groups of adjacent nucleotides. RNaseH hydrolyzes RNA strands of a RNA:DNA hybrid molecule.

Which of the following is not required for DNA replication by PCR? a. Oligonucleotide primers b. DNA polymerase c. DNA ligase d. Deoxynucleotides

C. DNA ligase is an enzyme that catalyzes the reaction between the 5'-phosphate end of one DNA fragment and the 3'-hydroxyl end of the next. This "nick sealing" requires energy from ATP hydrolysis, thus remaking the broken phosphodiester bond between the adjacent nucleotides. Ligase is a very important enzyme in DNA repair, but it is not used in a polymerase chain reaction (PCR). PCR does not require a DNA template, two primers to anneal to nucleotide sequences flanking the desired amplification sequence, deoxynucleotide triphosphates (dNTPs) to be used as building blocks for the growing DNA chain, DNA polymerase, and magnesium chloride as an essential cofactor for DNA polymerase activity.

Which of the following items is not used in the preparation of a DNA probe for Southern blotting using random hexamer primers? a. Template DNA b. Three unlabeled nucleotides c. Dideoxynucleotides, with one of them labeled d. DNA polymerase

C. Preparation of a DNA probe using random hexamer primers requires a DNA template containing the desired target sequence; four deoxynucleotides, at least one of which must be labeled (e.g., radionuclide, fluorescent, biotin, etc.); and DNA polymerase. The double-stranded DNA template (25-50 ng) is denatured, and a mixture of random oligonucleotides of six bases in length anneal to the template DNA. The primers are extended by the action of a DNA polymerase (e.g., T7 DNA polymerase) in the presence of one or two labeled deoxynucleotide triphosphates. Random primed probes are generally 500 nucleotides in length. Solutions containing the labeled probes are incubated with the blot. Hybridization of the labeled probe will occur if the gene being sought is present in the DNA on the blot to give a positive signal. Dideoxynucleotides are used in preparing samples for DNA sequencing by the Sanger method and cause DNA polymerization to cease.

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 (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 (RT-PCR) is a method that determines whether a gene is being expressed. The starting material for RT-PCR 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. 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.

TaqMan probes used to increase specificity of real-time PCR assays generate a fluorescent signal a. at the beginning of each cycle during the denaturation step b. when the probes bind to the template (i.e., during annealing) c. when the probe is digested by 5'->3' exonuclease activity during extension of primers (i.e., DNA synthesis) d. When the reporter fluorophor on the probe is separated from the quencher molecule by a restriction enzyme

C. Real-time PCR or quantitative PCR (qPCR) is a modification of PCR that allows quantification of input target sequences without addition of competitor templates or multiple internal controls. qPCR is used to measure copy numbers of diseased human genes and viral and tumor load and to monitor treatment effectiveness. The accumulation of double-stranded PCR products during PCR as they are generated can be measured by adding fluorescent dyes that are dsDNA-specific to the reaction mix, such as SYBR green. However, misprimed products or primer dimers will also generate fluorescence and give falsely high readings. Thus, more specific systems utilizing probes to generate signal, such as the TaqMan probes, Molecular Beacons, and Scorpion-type primers, have been developed. In the TaqMan probe-based system, specific primers are present to prime the DNA synthesis reaction catalyzed by Taq polymerase, thus forming the cDNA product. The TaqMan probe bings to a smaller region within the target sequence. The TaqMan probe has a 5' reporter fluorophor and 3' quencher molecule. During extension of the primers by Taq polymerase to form cDNA product (i.e., DNA synthesis), the 5'->3' exonuclease activity of Taq polymerase digests the TaqMan probe separating the reporter molecule from the quencher to generate a fluorescent signal. Molecular Beacon probes form hairpin structures due to short inverted repeat sequences at each end. The probe has a reporter dye at its 5' end and a quencher dye at its 3'. In the unbound state, fluorescence is suppressed because reporter and quencher dyes are bound closely together by the short inverted repeat sequences. In qPCR assays, fluorescence occurs when molecular beacon probe binds the denatured template during the annealing step because reporter dye is separated from the quencher molecule. Scorpion primers, which contain a fluorophore and a quencher, are covalently linked to the probe. In the absence of the target, the quencher absorbs fluorescence emitted by the fluorophore. During the PCR reaction, in the presence of the target, the fluorophore and the quencher of the Scorpion primers separate, resulting in an increase in the fluorescence emitted. All of these systems require excess concentrations of the labeled probe/primer, so fluorescence emitted is directly proportional to the amount of template available for binding.

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 2-4 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 from 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 fragine 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. 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 59. 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 MnlI. Primers used in this example generate a 223 bp PCR product from patient DNA. After resulting PCR products are digested with MnlI, normal patients product the following DNA fragments: 104 bp, 82 bp, 37 bp. In color plate 59, 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. Refer to color plate 59. Factor V Leiden mutation (A506G) causes activated protein C resistance that results in increased risk of hypercoagulability. The mutation destroys a MnlI 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 MnlI 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 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.

Which of the following is not an example of target amplification? a. Reverse transcription-PCR (RT-PCR) b. Transcription mediated amplification (TMA) c. Branched chain DNA amplification (bDNA) d. Polymerase chain reaction (PCR)

C. Target amplification refers to a process that increases the number of copies of the target DNA or RNA nucleotide sequence. Examples include the polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), transcription-mediated amplificaton (TMA), and nucleic acid sequence-based amplification (NASBA). Signal amplification will cause increased signal strength without increasing the number of target molecules. One example of signal amplification is the branched chain DNA reaction. Probe amplification will increase the number of copies of the probe that is complementary to the target. One example of probe amplification is the ligase chain reaction.

A restriction enzyme recognizes the sequence, 5' CT^ATAG 3', and cuts as indicated. Predict the ends that would result on the complementary DNA strand. a. 3' G 5' 3' ATATC 5' b. 3' GA 5' 3' TATC 5' c. 3' GATA 5' 3' TC 5' d. 3' GATAT 5' 3' C 5'

C. The complementary strand for this DNA sequence would be, read left to right, 3' GATATC 5'. Restriction endonucleases require dsDNA, because they use as their substrate palindromic molecules, meaning a molecule that will "read" the same left to right or right to left. In this instance, the complementary strand, read 5' to 3' (right to left), reads the same as the sense strand, read 5' to 3'. If the enzyme cuts the sense strand as indicated, between the thymine and adenine, it will cut the complementary strand identically. This will leave, on the sense strand, the two sequences 5' CT 3' and 5' ATAG 3'. The complementary strand will show 3' GATA 5' and 3' TC 5'. Check again to see that these new sequences actually are identical, read 5' to 3', on both strands.

The absorbance of a 1:100 dilution of isolated dsDNA solution, measured at 260 nm, is 0.062. What is a reasonable estimate for the dsDNA concentration of the sample, expressed in microg/mL? a. 3.1 b. 6.2 c. 310 d. 5000

C. The concentration of dsDNA can be estimated by taking its absorbance reading at 260 nm and multiplying that absorbance by a factor of 50, because one absorbance unit at 260 nm equals approximately 50 μg/mL. To solve this problem: 100 (dilution factor) x 0.062 (sample Abs. at 260 nm) x 50 μg/mL (conversion factor for dsDNA) = 310 μg/mL.

A 5850-base plasmid possesses EcoRI restriction enzyme cleavage sites at the following base pair locations: 36, 1652, and 2702. Following plasmid digestion, the sample is electrophoresed in a 2% agarose gel. A DNA ladder marker, labeled M in Color Plate 56, is included in the first lane, which base pair sizes indicated in lanes A through D. Which lane represents the sample pattern that is most likely the digested plasmid? a. A b. B c. C d. D

C. To solve this problem, it is necessary to recognize that plasmid DNA exists as a closed circle. This means that base pair #1 is adjacent to base pair #5850. If the enzyme cleaves the plasmid at positions 36, 1652, and 2702, this will result in three pieces of DNA. One piece will contain base pairs (bp) 37 through 1652 (with a size of 1616 bp), a second will contain bp 1653 through 2702 (with a size of 1050 bp), and the third will plan the sequence from bp 2703 through 5850 and from 1 to 36 (with a size of 3184 bp). Note that to determine the size of each piece, subtract the numbers corresponding to each adjacent cut site (e.g., 1652 - 36 = 1616 amd 2702 - 1652 = 1050). For the third piece, subtract the highest numbered cut site (i.e., 2702) from the total size of the plasmid (i.e., 5850), and add the size of the piece beginning at bp #1 through bp #36. Use the DNA ladder marker (lane M) in color plate 56 to predict the placement of these pieces (3184 bp, 1616 bp, and 1050 bp) of DNA on the final electrophoresis pattern.

In the presence of salt, DNA is precipitated from solution by a. 10 mM Tris, 1 mM EDTA b. 0.1% sodium dodecyl sulfate (SDS) c. Alkaline buffers, such as 0.2 N NaOH d. Alcohols, such as 95% ethanol or isopropanol

D. Alcohol precipitation of nucleic acids is a standard method in molecular biology. Sterile water, 10 mM Tris, 1 mM EDTA, or 0.1% SDS can be used to rehydrate DNA; 1 mM EDTA and 0.1% SDS are included in these mixes to inhibit DNases. Alkaline solutions, such as 0.2 N NaOH, are used to denature nucleic acids.

How many chromosomes are contained in a normal human somatic cell? a. 22 b. 23 c. 44 d. 46

D. DNA in human somatic cells is compartmentalized into 22 pairs of chromosomes, reffered to as autosomes. They are numbered 1 through 22. In addition, humas 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.

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 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.

Which of the following assays cannot be accomplished using PCR methods employing only Taq 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. Hepatitis C virus (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 considered a "high stringency" condition for DNA probe protocols? a. Using wash buffer with highly acidic pH b. Washing the matrix with high-salt buffer c. Radiolabeling the probe with 35S rather than 32P d. Washing the transfer membrane (e.g., nitrocellulose or nylon) at high temperature

D. In Southern blots, hybrids can form between molecules with similar but not necessarily identical sequences. The washing conditions used after adding the labeled probe can be varied so that hybrids with differing mismatch frequencies are controlled. The higher the wash temperature or the lower the salt concentration in the wash buffer, the higher the stringency. Increasing the stringency will decrease the number of mismatches that form between the probe and the target DNA.

Which of the following specimen types is not used routinely as source material for molecular genetic tests? a. Whole blood b. Buccal scrapings c. Amniocytes d. Rectal swabs

D. Many frequently used protocols in molecular biology involve PCR. Several substances can inhibit this reaction. For example, because of the nature of fecal material, it is not routinely used, and materials in swabs have also been reported to inhibit PCR. Therefore, a more appropriate specimen that could be used for PCR would be a stool filtrate. Nucleated cells are necessary for isolation of DNA. Whole blood is an acceptable specimen. White blood cells are the source of DNA in this type of specimen and must be separated from red blood cells as soon as possible because hemoglobin will inhibit PCR. For diagnosis of blood parasites, such as Babesia and Plasmodium, a hemolyzed and washed red blood cell sample is preferred for recovery of the DNA from the parasites. Amniocytes are used for molecular cytogenetic testing to prenatally screen for genetic diseases. Noninvasive collection of cells for genetic and forensic testing can be obtained from the buccal (oral) mucosa.

In RNA, which nucleotide base replaces thymine of DNA? a. adenine b. cytosine c. guanine 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.

The technique that makes ssDNA from an RNA template is called a. Strand displacement amplification b. Polymerase chain reaction c. Ligase chain reaction d. Reverse transcription

D. The process whereby a strand of RNA is synthesized from template DNA is called transcription. The enzyme involved is RNA polymerase. It is possible, however, as retroviruses have shown, to produce DNA using template RNA. This reversal of the central nucleic acid dogma is called "reverse transcription," and the enzyme that performs this is called reverse transcriptase. After synthesizing a single-stranded DNA molecule from RNA, a different enzyme (DNA polymerase) then synthesizes a complementary strand to produce a DNA double helix. The other three answers describe amplification methods designed to increase the sensitivity of molecular diagnostic tests. They accomplish this by making copies of either the target nucleic acids (e.g., PCR), or the probe molecules (e.g., ligase chain reaction and strand displacement amplification), or the signal produced (e.g., branched chain DNA reaction).

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. The translocation resulting in the Philadelphia chromosome can be detected by reverse transcription-polymerase chain reaction (RT-PCR), Southern blot, and cytogenetic analysis. The presence of a Philadelphia (Ph) chromosome confirms the diagnosis of chromic 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 22q11. 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 BCR/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.