Gel Electrophoresis in Nucleic Acid Analysis

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Questions and Answers

What is the primary function of the CRISPR-associated protein (Cas) enzyme?

To recognize folded nucleic acids

To cleave the invading DNA at specific locations (correct)

To guide the Cas enzyme to the matching invading DNA

To transcribe short RNA sequences from the CRISPR spacer regions

What is the main advantage of CRISPR/Cas9 over traditional restriction enzymes?

CRISPR/Cas9 can recognize folded nucleic acids

CRISPR/Cas9 can only be used for DNA analysis

CRISPR/Cas9 can only cleave single-stranded nucleic acids

CRISPR/Cas9 provides the specificity of restriction enzymes with the versatility of guiding cuts to any sequence site (correct)

What is the primary purpose of the Southern blot?

To analyze RNA structure and expression

To analyze specific DNA regions in a complex genome by RFLP (correct)

To screen for mutations and polymorphisms

To detect proteins and protein modifications

What is the primary goal of genomics?

To analyze hundreds to thousands of targets or whole genomes Signup and view all the answers

What is the purpose of the spacer sequences in the CRISPR system?

To serve as adaptive immunity with memory of the invading DNA Signup and view all the answers

What is the primary application of CRISPR/Cas9 in the laboratory?

To alter DNA at user-defined locations Signup and view all the answers

What is the main advantage of CRISPR RNA over traditional restriction enzymes?

CRISPR RNA can lead transcription activators, repressors, gene promoters, or reporter molecules to target sequences Signup and view all the answers

What is the purpose of a nucleating agent in liquid acrylamide solutions?

To facilitate the solidification of acrylamide Signup and view all the answers

What is the primary function of a density agent in a loading solution?

To facilitate the loading of the sample into the wells Signup and view all the answers

What is the purpose of a fluorescent stain in agarose gel electrophoresis?

To visualize the nucleic acid fragments Signup and view all the answers

What is the primary advantage of capillary electrophoresis over agarose gel electrophoresis?

Increased sensitivity Signup and view all the answers

What is the primary purpose of comparative genomic hybridization arrays?

To detect amplifications or deletions in DNA Signup and view all the answers

What type of array is used to measure gene expression levels?

RNA expression arrays Signup and view all the answers

What is the function of a platinum wire in agarose gel electrophoresis?

To establish a current through the gel Signup and view all the answers

What is the term for the process of detecting chromosome microdeletions using microarrays?

Virtual karyotyping Signup and view all the answers

What is the purpose of a molecular weight standard in agarose gel electrophoresis?

To estimate the size of the DNA fragments Signup and view all the answers

What is the primary difference between agarose gel electrophoresis and capillary electrophoresis?

The presence of a gel matrix Signup and view all the answers

What is the purpose of labeling the sample material in microarray analysis?

To allow for hybridization of the sample to the probes Signup and view all the answers

What is the purpose of a laser in capillary electrophoresis?

To excite the fluorescent labels Signup and view all the answers

What is the term for the type of array that can be used to determine DNA nucleotide sequence?

High-density oligonucleotide arrays Signup and view all the answers

What is the purpose of using a reference material in microarray analysis?

To compare the levels of gene expression between the test and reference samples Signup and view all the answers

What is the advantage of using multiple capillaries in capillary electrophoresis?

Ability to analyze multiple samples simultaneously Signup and view all the answers

What is the purpose of a visual dye in a loading solution?

To visualize the sample during electrophoresis Signup and view all the answers

What is the term for the type of array that uses beads as the solid support?

Bead array Signup and view all the answers

What is the purpose of using fluorescent labels in bead array assays?

To detect the presence of the target protein or nucleic acid Signup and view all the answers

What is the advantage of bead array assays over other types of arrays?

They can detect multiple targets simultaneously Signup and view all the answers

What is a clinical application of bead array assays?

HLA typing Signup and view all the answers

What is the primary function of restriction enzymes in DNA analysis?

To separate phosphodiester bonds between nucleotides Signup and view all the answers

What is the purpose of molecular weight standards in capillary gel electrophoresis?

To enable instrument to automatically assess fragment size Signup and view all the answers

What is the primary mechanism of DNA separation in capillary gel electrophoresis?

Electrokinetic process Signup and view all the answers

What is the function of CRISPR-Cas9 system in DNA analysis?

To recognize and bind to specific nucleotide sequences Signup and view all the answers

What is the purpose of formamide in preparing DNA samples for capillary gel electrophoresis?

To denature DNA into single strands Signup and view all the answers

What is the result of DNA digestion with restriction enzymes?

Production of DNA fragments of varying sizes Signup and view all the answers

What is the characteristic of DNA fragments produced by restriction enzymes?

Fragments are specific to the DNA sequence Signup and view all the answers

What is the purpose of nucleic acid tests?

To detect changes in DNA sequence Signup and view all the answers

What is the name of the first restriction enzyme isolated from E. coli?

EcoR1 Signup and view all the answers

What is the primary application of restriction fragment length polymorphism (RFLP) analysis?

All of the above Signup and view all the answers

What is the primary purpose of electrophoresis in the analysis of nucleic acids?

To observe the sizes or amounts of nucleic acid Signup and view all the answers

Which type of gel is more suitable for separating very large nucleic acids of tens of thousands of base pairs?

Agarose gel at low concentrations Signup and view all the answers

Why do smaller nucleic acid chains move faster through the gel matrix during electrophoresis?

Because they have a smaller mass-to-charge ratio Signup and view all the answers

What is the purpose of using a standard molecular weight marker in gel electrophoresis?

To estimate the size in bases or base pairs of the test nucleic acids Signup and view all the answers

Which of the following is a characteristic of polyacrylamide gels?

More precisely designed for high-resolution separation Signup and view all the answers

During electrophoresis, which pole do negatively charged nucleic acids move towards?

The anode Signup and view all the answers

What determines the proper type and concentration of gel to be used in electrophoresis?

The expected sizes of the nucleic acids to be separated Signup and view all the answers

Why are agarose gels less expensive and less toxic to use than polyacrylamide gels?

Because they are natural polymers Signup and view all the answers

What is the primary function of oligonucleotide primers in a PCR reaction?

To ensure the specificity of the PCR reaction Signup and view all the answers

What is the typical temperature range for the annealing step in a standard PCR cycle?

50°C–70°C Signup and view all the answers

What is the purpose of the initial 5- to 15-minute incubation at the denaturation temperature in some PCR protocols?

To activate specialized DNA polymerases Signup and view all the answers

What is the typical length of PCR products?

50 to more than 1,000 bp Signup and view all the answers

What is the purpose of the final 7- to 10-minute step at the extension temperature in some PCR protocols?

To ensure complete copying of the products Signup and view all the answers

What is the effect of ramp speed on PCR amplification?

It increases the efficiency of the amplification Signup and view all the answers

What is the purpose of the denaturation step in a standard PCR cycle?

To separate the double-stranded DNA into single strands Signup and view all the answers

What is the instrument used to carry out the amplification program in PCR?

Thermal cycler Signup and view all the answers

What is the typical number of cycles in a PCR amplification program?

20 to 50 cycles Signup and view all the answers

At what temperature is the extension step typically carried out in a standard PCR cycle?

68°C–72°C Signup and view all the answers

What is the primary purpose of the internal amplification control in qPCR and RT-qPCR?

To verify that negative results are true negatives and not due to amplification failure Signup and view all the answers

What is the primary difference between qPCR and digital droplet PCR?

The method of quantification of the target DNA Signup and view all the answers

What is the purpose of the limiting dilution of sample template molecules in digital droplet PCR?

To prepare individual droplets of reaction buffer in oil Signup and view all the answers

What is the primary application of qPCR in the detection of microorganisms?

To detect microorganisms that are difficult or dangerous to culture in the laboratory Signup and view all the answers

What is the function of the fluorescent dye in the capillary gel electrophoresis detection of PCR products?

To label the PCR primer Signup and view all the answers

What is the advantage of multiplex qPCR methods?

To assess multiple targets simultaneously Signup and view all the answers

What is the primary difference between the lag phase and the log phase in the PCR curve?

The rate of PCR product accumulation Signup and view all the answers

What is the purpose of the standard curve in qPCR?

To convert the Ct value to the number of DNA copies in the starting sample Signup and view all the answers

What is the primary advantage of digital droplet PCR over qPCR?

It provides absolute quantification Signup and view all the answers

What is the function of the emulsion formed in digital droplet PCR?

To act as individual reaction chambers Signup and view all the answers

What is the primary purpose of gel or capillary gel electrophoresis in a PCR procedure?

To visualize the products of the PCR reaction Signup and view all the answers

What is the role of reverse transcriptase in RT-PCR?

To synthesize cDNA from RNA Signup and view all the answers

What is the purpose of the 3′ end of the primer in SSP-PCR?

To anneal to the template on a potentially mutated or polymorphic base pair Signup and view all the answers

What is the advantage of using probes in qPCR compared to SYBR green?

Probes provide higher specificity for the intended product Signup and view all the answers

What is the purpose of the 5′ end of the primer in PCR?

To allow for the attachment of noncomplementary sequences containing restriction enzyme recognition sites Signup and view all the answers

What is the difference between RT-PCR and qPCR?

RT-PCR is used for qualitative detection, while qPCR is used for quantitative detection Signup and view all the answers

What is the purpose of FRET, TaqMan, molecular beacon, and scorpion probes in qPCR?

To hybridize to the template by sequence complementarity and generate fluorescent signals Signup and view all the answers

What is the advantage of using qPCR compared to standard PCR?

qPCR allows for the quantification of starting material Signup and view all the answers

What is the purpose of SYBR green in qPCR?

To detect the accumulation of PCR product in real time Signup and view all the answers

What is the purpose of sequence-specific primer PCR (SSP-PCR)?

To detect mutations and polymorphisms Signup and view all the answers

What is the primary function of RNase II in the process of probe amplification?

To degrade hybridized RNA, leaving single-stranded DNA Signup and view all the answers

In the process of strand displacement amplification, what is the function of the outer primer?

To bind to the target DNA and extend, displacing the product formed by the simultaneous extension of the inner primer Signup and view all the answers

What is the primary advantage of loop-mediated isothermal amplification (LAMP)?

Ability to amplify target DNA in a short amount of time Signup and view all the answers

In molecular inversion probe (MIP) amplification, what happens to the probe ends when they bind to target sequences?

They are brought together and ligated to form circles Signup and view all the answers

What is the primary function of branched DNA (bDNA) amplification?

To capture the target nucleic acid and bind to multiple reporter molecules Signup and view all the answers

What is the main advantage of branched DNA (bDNA) amplification over methods using a single probe or primer?

Higher specificity due to the use of multiple probes Signup and view all the answers

What is the primary function of DNA?

To store genetic information Signup and view all the answers

What is the primary difference between probe amplification and signal amplification?

Probe amplification amplifies the target DNA sequence, while signal amplification amplifies the signal bound to the target Signup and view all the answers

What is the purpose of the restriction enzyme in strand displacement amplification (SDA)?

To form a nick (cutting only one strand of the double helix) in the double-stranded DNA segment Signup and view all the answers

Which of the following sequencing methods uses modified nucleotide bases called dideoxynucleotide triphosphates (ddNTPs)?

Chain termination sequencing Signup and view all the answers

What is the primary application of molecular inversion probe (MIP) amplification?

All of the above Signup and view all the answers

What is the result of the sequencing reaction in Sanger sequencing?

A collection of fragments of various sizes Signup and view all the answers

What is the advantage of using multiple probes in branched DNA (bDNA) amplification?

Enhanced specificity due to the use of multiple probes Signup and view all the answers

What is the purpose of using fluorescent labels in Sanger sequencing?

To distinguish between the different ddNTPs Signup and view all the answers

What is the purpose of resolving the DNA ladder by gel or capillary gel electrophoresis?

To separate the DNA fragments based on their size Signup and view all the answers

What is the term for the collection of fluorescent peaks obtained by capillary gel electrophoresis?

Electropherogram Signup and view all the answers

What is the purpose of the primer pair in Sanger sequencing?

To outline the target DNA Signup and view all the answers

What is the reason why synthesis will stop if a ddNTP is incorporated into the growing DNA chain?

Because the ddNTP lacks an OH group at the 3′ carbon of the deoxyribose sugar Signup and view all the answers

What is the advantage of capillary electrophoresis over gel electrophoresis?

It is faster and more automated Signup and view all the answers

What is the purpose of bDNA signal amplification assay?

To detect and quantify hepatitis B virus, HCV, and HIV-1 Signup and view all the answers

What is the primary purpose of including positive and negative controls in in situ hybridization (ISH) testing?

To ensure the accuracy of the results and verify tissue preparation Signup and view all the answers

What is the main function of programmed cell death ligand (PD-L1) in the immune response?

To suppress the adaptive immune response by binding to programmed cell death protein 1 (PD-1) on activated lymphocytes and dendritic cells Signup and view all the answers

What is the primary difference between fluorescence in situ hybridization (FISH) and in situ hybridization (ISH) methods?

FISH requires specialized microscopes equipped to detect fluorescent signals, whereas ISH does not Signup and view all the answers

What is the purpose of using reference probes in fluorescence in situ hybridization (FISH) methods?

To identify the chromosomes of interest while assessing deletion or amplification Signup and view all the answers

What is the term used to describe the sensitivity of in situ hybridization (ISH) methods to the buffer and temperature conditions of hybridization?

Stringency Signup and view all the answers

What is the main advantage of using array methods in molecular diagnostics?

They can detect multiple or complex genetic abnormalities Signup and view all the answers

What is the primary purpose of amplification methods in molecular diagnostics?

To copy nucleic acids for further analysis Signup and view all the answers

Who is credited with the development of the in vitro PCR, which greatly facilitated and broadened the potential applications of gene amplification?

Kerry Mullis Signup and view all the answers

What is the primary purpose of using immunohistochemistry in in situ hybridization (ISH) testing?

To detect the presence of clinically significant protein targets, such as those expressed by tumor cells Signup and view all the answers

What is the term used to describe the process of detecting targets in place as they appear in tissues, cells, and subcellular structures?

In situ hybridization (ISH) Signup and view all the answers

What is the primary mechanism by which TaqMan probes generate a signal?

The target DNA is copied, releasing the reporter dye from the quencher. Signup and view all the answers

What is the key difference between molecular beacons and TaqMan probes?

Molecular beacons have a quencher and a reporter dye, but they are not separated in the presence of target sequences. Signup and view all the answers

What is the purpose of the scorpion probe?

To generate a signal as the target DNA is copied, with the reporter covalently attached to the product. Signup and view all the answers

What is the primary advantage of digital droplet PCR over qPCR?

It is more sensitive and can detect smaller changes in target number. Signup and view all the answers

What is the primary mechanism of transcription-based amplification systems?

A complementary DNA copy is synthesized from the target RNA, and then transcription of the cDNA produces millions of copies of RNA products. Signup and view all the answers

What is the primary advantage of targeting RNA in transcription-based amplification systems?

It is more sensitive than targeting DNA because each microorganism makes multiple copies of RNA. Signup and view all the answers

What is the primary application of transcription-mediated amplification (TMA)?

Detection of RNA viruses, such as HCV and HIV. Signup and view all the answers

What is the purpose of the cDNA:RNA hybrid in transcription-mediated amplification?

To form a template for the synthesis of more cDNA. Signup and view all the answers

What is the advantage of TMA over PCR?

TMA is an isothermal process, which does not involve the repeated heating and cooling required for PCR. Signup and view all the answers

What is the primary application of digital PCR in infectious disease?

Detection of rare mutations in infectious diseases. Signup and view all the answers

What is the main purpose of Sanger sequencing in genetics and oncology?

To identify gene abnormalities in inherited diseases Signup and view all the answers

Why is pyrosequencing less sensitive than other methods for detecting DNA sequence changes?

Because it is affected by the heterozygous form of the alteration Signup and view all the answers

What is the purpose of sequencing the complementary strand of the DNA in Sanger sequencing?

To confirm the alteration detected in the original sequence Signup and view all the answers

What is the advantage of pyrosequencing over Sanger sequencing?

It is less labor-intensive and more convenient for short sequence analyses Signup and view all the answers

What is the purpose of the sequencing primer in pyrosequencing?

To initiate the DNA synthesis reaction Signup and view all the answers

What is the result of the pyrosequencing reaction?

A pyrogram of luminescent peaks Signup and view all the answers

Why is pyrosequencing used in genetics and oncology?

To sequence targeted areas of known gene locations Signup and view all the answers

What is the limitation of pyrosequencing compared to Sanger sequencing?

It produces shorter sequence reads Signup and view all the answers

What is the purpose of adenosine 5′ phosphosulfate (APS) in pyrosequencing?

To convert pyrophosphate to ATP Signup and view all the answers

What is the advantage of using some instruments developed for genomic or next-generation sequencing (NGS) in pyrosequencing?

They utilize the pyrosequencing chemistry Signup and view all the answers

What was the primary goal of developing Next-Generation Sequencing (NGS) technologies?

To sequence the human genome for a minimal cost of less than $1,000 Signup and view all the answers

What is a characteristic of NGS procedures?

Starting with short DNA templates, usually less than 500 bp Signup and view all the answers

Which of the following NGS technologies has been most frequently applied to clinical applications?

Pyrosequencing, reversible dye sequencing, and ion conductance sequencing Signup and view all the answers

What was the primary challenge that was not initially included in the sequencing cost of NGS technologies?

The cost of interpreting the data Signup and view all the answers

What is the primary difference between NGS and traditional Sanger sequencing?

The number of genomes that can be sequenced simultaneously Signup and view all the answers

What is the term for the goal of sequencing the human genome for a minimal cost of less than $1,000?

The $1,000 genome Signup and view all the answers

What is the primary benefit of using large databases of somatic variant data in cancer diagnosis and treatment?

To refine interpretation of newly collected data Signup and view all the answers

What is the primary advantage of NGS technologies over traditional Sanger sequencing?

Lower cost of sequencing Signup and view all the answers

What is the name of the project that has sequenced thousands of genomes using NGS technologies?

The 1,000 Genome Project Signup and view all the answers

What is the primary advantage of using NGS over Sanger sequencing?

NGS provides higher coverage of regions of interest Signup and view all the answers

What is the purpose of using quality, variant type, and allele frequency parameters in filtering?

To identify the most biologically significant variants Signup and view all the answers

Why is adequate coverage of regions of interest required in NGS?

To provide sufficient data for confident base calling Signup and view all the answers

What is the primary purpose of annotating variants using historical sequence database information?

To determine the medical significance of variants Signup and view all the answers

What is the primary benefit of using a standard nomenclature system for sequence information?

To facilitate clear communication and organized storage of sequence data Signup and view all the answers

What is the primary purpose of using targeted gene panels in NGS?

To produce a finished report with variants identified automatically Signup and view all the answers

Why are errors more common in NGS compared to Sanger sequencing?

Library preparation is more error-prone in NGS Signup and view all the answers

What is the primary purpose of using FASTQ files in NGS?

To include a quality symbol for each base Signup and view all the answers

What is the primary benefit of using bioinformatics in molecular analyses?

To facilitate the interpretation of large amounts of data Signup and view all the answers

What is the primary purpose of comparing the sequence of a DNA sample to a reference sequence in NGS?

To identify variants or polymorphisms in the sample Signup and view all the answers

What is the minimum coverage required to call rare variants in NGS?

500X Signup and view all the answers

What is the primary application of targeted NGS in the clinical laboratory?

HLA typing, oncology, and genetics Signup and view all the answers

What is the purpose of the Cancer Genome Atlas (TCGA) program?

To understand the genetic basis of cancer and improve diagnosis and treatment Signup and view all the answers

What is the term for the number of mutations per megabase of sequenced DNA in a tumor?

Tumor mutational burden Signup and view all the answers

What is the primary advantage of NGS over other sequencing technologies?

Ability to investigate all known genetic loci Signup and view all the answers

What is the purpose of sequencing gene panels in NGS?

To identify genetic variants in a specific set of genes Signup and view all the answers

What is the term for the percentage of sequences carrying a variant in NGS?

Allele frequency Signup and view all the answers

What is the primary advantage of NGS in HLA typing?

Ability to resolve ambiguities Signup and view all the answers

What is the primary purpose of using PCR primers or probes in NGS?

To select and copy targeted regions for sequencing Signup and view all the answers

What is the primary factor that limits the degree of coverage used in NGS?

Cost Signup and view all the answers

What is the function of the bar code or index in NGS?

To identify each sample and gene region in the library Signup and view all the answers

What is the purpose of emulsion PCR in NGS?

To generate a library of PCR products attached to beads Signup and view all the answers

What is the mechanism of detection in ion conductance sequencing?

Release of hydrogen ions and detection of pH change Signup and view all the answers

What is the purpose of bridge PCR in reversible dye terminator technology?

To form clusters of templates across the flow cell Signup and view all the answers

What is the advantage of ion conductance sequencing over other methods?

It is faster and does not require optical sensing Signup and view all the answers

What is the purpose of adapters in NGS?

To carry PCR primer-binding sites and allow unbiased amplification Signup and view all the answers

What is the purpose of the sequencing reagents in pyrosequencing?

To release PPi and generate light signals Signup and view all the answers

What is the advantage of using multiple samples in a single run of NGS?

It decreases the cost and time required for sequencing Signup and view all the answers

What is the purpose of the picoplate in pyrosequencing?

To carry out independent sequencing reactions in hundreds of thousands of wells Signup and view all the answers

What is the primary function of flow cytometry in the clinical laboratory?

To monitor HIV infection status and enumerate hematopoietic stem cells Signup and view all the answers

What is the primary purpose of using fluorescent-labeled antibodies or probes in flow cytometry?

To identify specific surface or cytoplasmic proteins Signup and view all the answers

What is a characteristic of fluorochromes used in flow cytometry?

They absorb light across a spectrum of wavelengths and emit light of lower energy across a spectrum of longer wavelengths Signup and view all the answers

What is the primary application of flow cytometry in the diagnosis of diseases?

In the diagnosis of leukemia and lymphoma Signup and view all the answers

What is the primary advantage of using multiple fluorochromes in flow cytometry?

It allows for the analysis of multiple cell properties simultaneously Signup and view all the answers

What is the primary function of laser light in flow cytometry?

To excite fluorochromes and detect cell properties Signup and view all the answers

What is the primary application of flow cytometry in transplantation?

To detect human leukocyte antigen (HLA) antibodies Signup and view all the answers

What is the primary advantage of using flow cytometry in cell analysis?

It allows for the analysis of a large number of cells simultaneously Signup and view all the answers

What is the primary purpose of using optical filters in flow cytometry?

To maximize collection of light from specific fluorochromes Signup and view all the answers

What is the effect of using Ficoll-Hypaque density gradient centrifugation on some cell populations?

Selective loss of some cell populations Signup and view all the answers

What is the purpose of using erythrocyte lysis techniques in sample preparation for flow cytometry?

To destroy erythrocytes while leaving WBCs intact Signup and view all the answers

What is the typical range of digital signals measured in flow cytometry?

1 to 256 channels Signup and view all the answers

What is the purpose of using a marker in flow cytometry data analysis?

To differentiate cells with low levels of fluorescence from cells with high levels of fluorescence Signup and view all the answers

What is the primary purpose of using fiber-optic cables in flow cytometry?

To direct light to the photomultiplier tubes Signup and view all the answers

What is the effect of using heparin as an anticoagulant in sample preparation for flow cytometry?

Improved stability of samples for up to 48 hours Signup and view all the answers

What is the primary purpose of using EDTA as an anticoagulant in sample preparation for flow cytometry?

To collect whole blood samples for analysis within 30 hours Signup and view all the answers

What is the typical number of events collected for each sample in flow cytometry data acquisition?

10,000 to 20,000 events Signup and view all the answers

What is the primary purpose of using a single-parameter histogram in flow cytometry data analysis?

To plot a chosen parameter versus the number of events Signup and view all the answers

What is the primary advantage of flow cytometry?

It enables the accurate detection of cells that are present in very small numbers. Signup and view all the answers

What is the purpose of the fluidics system in a flow cytometer?

To ensure that cells pass through the laser light one cell at a time. Signup and view all the answers

What determines which fluorochromes can be used in an assay?

The wavelength of the laser light. Signup and view all the answers

What is the primary defect in patients with paroxysmal nocturnal hemoglobinuria (PNH)?

Lack of GPI anchor proteins on the cell surface Signup and view all the answers

Which of the following tests can detect minimal residual disease (MRD) in patients with leukemia or lymphoma?

Flow cytometry Signup and view all the answers

What is the difference between forward scatter and side scatter?

Forward scatter measures size, while side scatter measures granularity. Signup and view all the answers

What is the purpose of using fluorescent-labeled antibodies in flow cytometry?

To detect the presence of specific antigens on the surface of cells. Signup and view all the answers

What is the primary application of flow cytometry in transplant immunology?

HLA typing and cross-matching Signup and view all the answers

What is the advantage of using multiple lasers in a flow cytometer?

It enables the analysis of up to five fluorochromes at a time. Signup and view all the answers

What is the advantage of using monoclonal antibodies over polyclonal antibodies in immunophenotyping?

Monoclonal antibodies are more specific Signup and view all the answers

What is the primary function of cytometric bead arrays?

Identifying multiple analytes simultaneously Signup and view all the answers

What is the purpose of the optics and photodetectors in a flow cytometer?

To detect the light scatter and fluorescence signals generated by the cells. Signup and view all the answers

What is the term for the parameters that are intrinsic to the cell?

Intrinsic parameters. Signup and view all the answers

Which of the following is an application of flow cytometry in molecular diagnostics?

Detecting viral nucleic acids Signup and view all the answers

What is the advantage of using multicolor analysis in flow cytometry?

It enables the analysis of multiple extrinsic parameters simultaneously. Signup and view all the answers

What is the primary advantage of flow cytometry over traditional serological methods?

Faster and more accurate results Signup and view all the answers

What is the purpose of analyzing the data generated by the flow cytometer in conjunction with forward scatter and side scatter?

To enable more accurate subtyping of cells. Signup and view all the answers

What is the purpose of fluorescent labels in bead array assays?

To detect analytes Signup and view all the answers

Which of the following is a clinical application of flow cytometry?

All of the above Signup and view all the answers

What is the primary purpose of a gate in a dual-parameter dot plot?

To filter out debris and isolate subpopulations of cells of interest Signup and view all the answers

What is the primary advantage of using flow cytometry in DNA analysis?

All of the above Signup and view all the answers

What is the advantage of using a single-platform analysis over a dual-platform analysis?

It eliminates the potential for added error associated with the use of two distinct methods Signup and view all the answers

What is the primary purpose of immunophenotyping in the clinical laboratory?

To characterize and enumerate normal lymphocytes Signup and view all the answers

What is the primary difference between quadrants 1 and 2 in a dual-parameter dot plot?

Quadrant 1 consists of cells that are positive for fluorescence on the y-axis and negative for fluorescence on the x-axis, while quadrant 2 consists of cells that are positive for fluorescence on both the x- and y-axes Signup and view all the answers

What is the primary purpose of the computer and specialized software in a dual-parameter dot plot analysis?

To calculate the percentage of cells in each quadrant Signup and view all the answers

What is the primary advantage of using bead-based methods over volumetric methods in single-platform analysis?

Bead-based methods provide a more accurate absolute count of a particular cell type Signup and view all the answers

What is the primary purpose of detailed phenotypic analysis in the clinical laboratory?

To determine the degree of differentiation and activation of a specific cell population Signup and view all the answers

What is the primary difference between nonmalignant immunophenotyping and malignant immunophenotyping?

Nonmalignant immunophenotyping is used to characterize and enumerate normal lymphocytes, while malignant immunophenotyping is used to diagnose malignant disorders Signup and view all the answers

What is the primary purpose of using a known quantity of fluorescent beads in a bead-based method?

To provide a simple mathematic calculation to directly determine the absolute WBC numbers Signup and view all the answers

What is the primary advantage of using a single-platform analysis over a traditional method?

It eliminates the potential for added error associated with the use of two distinct methods Signup and view all the answers

What is the primary purpose of immunophenotyping by flow cytometry in leukemia and lymphoma management?

To identify the lineage of hematopoietic malignancies Signup and view all the answers

Which of the following markers is typically paired with CD20 in the analysis of chronic lymphocytic leukemia (CLL)?

CD5 Signup and view all the answers

What is the significance of CD45 expression levels in flow cytometry analysis?

It is used to differentiate various populations of WBCs Signup and view all the answers

What is the primary application of flow cytometry in the evaluation of HIV disease?

To classify stages of HIV disease and determine treatment options Signup and view all the answers

What is the characteristic of neutrophils from patients with chronic granulomatous disease (CGD)?

They are unable to oxidize the dye dihydrorhodamine 123 Signup and view all the answers

What is the purpose of examining the FSC and SSC plot in flow cytometry analysis?

To ensure that a population is not being missed Signup and view all the answers

What is the significance of CD4+ T-cell numbers in HIV-infected patients?

They are used to classify stages of HIV disease and determine treatment options Signup and view all the answers

What is the primary purpose of immunophenotyping in the evaluation of immunodeficiency?

To compare patient values with reference ranges Signup and view all the answers

What is the significance of CD8+ T-cell levels in HIV-infected patients?

They are expanded during the early course of HIV disease Signup and view all the answers

What is the characteristic of blasts in terms of CD45 expression?

They have low levels of CD45 expression Signup and view all the answers

What was the primary motivation for the development of immunoassay analyzers?

To create an automated system capable of reducing or eliminating manual tasks Signup and view all the answers

What is a benefit of immunoassay automation in addition to reducing error and increasing accuracy?

The ability to provide more services with less staff Signup and view all the answers

What is a challenge of selecting an immunoassay analyzer for a laboratory?

The difficulty of determining which analyzer is best suited for a laboratory's needs Signup and view all the answers

What is a benefit of immunoassay automation in terms of reagent management?

The longer shelf life of reagents Signup and view all the answers

What is a common type of analyte that can be measured using immunoassay automation?

Low-level peptides such as peptide hormones Signup and view all the answers

How many different types of automated immunoassay analyzers are available?

More than 60 Signup and view all the answers

What is a consequence of eliminating manual steps in immunoassay testing?

The decrease in the likelihood of error Signup and view all the answers

What is a potential benefit of immunoassay automation in terms of sample management?

Sample delivery with bar codes for better sample identification Signup and view all the answers

What is a primary limitation of batch analyzers?

They can only perform one type of analysis at a time Signup and view all the answers

What is the purpose of clot detectors in stainless-steel probes?

To reject samples with clots Signup and view all the answers

What is a common issue associated with reusable pipette probes?

Carryover or contamination of samples Signup and view all the answers

What is the primary purpose of reagent storage compartments in some analyzers?

To store reagents at a controlled temperature Signup and view all the answers

What is the purpose of magnetic stirring in analyzers?

To mix samples with reagents Signup and view all the answers

What is the primary purpose of timed incubation in analyzers?

To incubate samples at a controlled temperature Signup and view all the answers

What is the primary advantage of using batch analyzers in clinical laboratories?

Cost-effectiveness when performing a large scale of a single type of testing Signup and view all the answers

What is the primary advantage of random-access analyzers over batch analyzers?

They can perform multiple analyses on a single sample Signup and view all the answers

What is the purpose of validating new instrumentation or methodology in clinical laboratories?

To meet the Clinical Laboratory Improvement Amendments (CLIA) regulations Signup and view all the answers

What is the primary purpose of fluorescent detectors in analyzers?

To measure the concentration of analytes in a sample Signup and view all the answers

What is the primary purpose of automation in analyzers?

To reduce the need for manual sampling Signup and view all the answers

Which of the following is a required verification for a new method in clinical laboratories?

All of the above Signup and view all the answers

What is the primary purpose of liquid-level sensors in analyzers?

To detect the lack of sample in a tube Signup and view all the answers

What is the definition of analytic specificity in clinical laboratory testing?

The ability to generate a negative result when the analyte is not present Signup and view all the answers

What is the purpose of parallel testing with an alternative method or technology in clinical laboratory testing?

To assess the accuracy of an assay Signup and view all the answers

What is the advantage of using random-access analyzers in clinical laboratories?

Flexibility in rapid processing of stat samples Signup and view all the answers

What is the purpose of the Centers for Medicare and Medicaid Services (CMS) in relation to clinical laboratory testing?

To provide an overview of the Clinical Laboratory Improvement Amendments (CLIA) regulations Signup and view all the answers

What is the definition of accuracy in clinical laboratory testing?

The ability to measure what the test claims to measure Signup and view all the answers

What is the purpose of calculating the standard deviation and coefficient of variation in clinical laboratory testing?

To evaluate the precision of an assay Signup and view all the answers

What is the purpose of including at least one normal and one abnormal control in the analysis of precision in clinical laboratory testing?

To ensure the precision of an assay is determined under different conditions Signup and view all the answers

Which of the following is NOT a nitrogen base found in nucleic acids?

Methionine (M) Signup and view all the answers

What is the primary function of DNA polymerase enzyme?

To catalyze the synthesis of DNA Signup and view all the answers

What is the term for a change in the nucleotide sequence of DNA?

Mutation Signup and view all the answers

What is the primary difference between DNA and RNA?

DNA is mostly double-stranded, while RNA is often single-stranded Signup and view all the answers

What is the purpose of a primer in DNA replication?

To provide the necessary 3' hydroxyl group for DNA replication Signup and view all the answers

What is the process by which genetic information flows from DNA to mRNA?

Transcription Signup and view all the answers

What is the result of a change in the nucleotide sequence of DNA, depending on its frequency?

A mutation or a polymorphism Signup and view all the answers

What is the purpose of RNA polymerase enzyme in RNA synthesis?

To catalyze the synthesis of RNA Signup and view all the answers

What is the main difference between capillary electrophoresis and agarose gel electrophoresis?

The type of gel used Signup and view all the answers

What is the primary purpose of hybridization in microarray analysis?

To bind two complementary strands of nucleic acids Signup and view all the answers

What is the main advantage of microarray analysis over other methods?

It can detect expression of thousands of genes simultaneously Signup and view all the answers

What is the primary function of restriction enzymes in DNA analysis?

To isolate specific parts of a DNA sequence for analysis Signup and view all the answers

What is the purpose of fluorescent labels in microarray analysis?

To aid in the detection of hybridized DNA Signup and view all the answers

What is the main limitation of microarray analysis?

It is expensive and not efficient compared to auto sequencing Signup and view all the answers

What is the primary application of microarray analysis?

Biomedical research, clinical diagnosis, and pharmaceuticals Signup and view all the answers

What is the purpose of submitting microarray data to the Gene Expression Omnibus Server?

To make the data available to researchers worldwide Signup and view all the answers

What is the primary mechanism of DNA separation in electrophoresis?

Charge of the DNA molecules Signup and view all the answers

What is the purpose of normalization in microarray data analysis?

To adjust for differences in intensity Signup and view all the answers

What is the primary function of the dichroic mirror in a fluorescence microscope?

To reflect light only at the excitation wavelength Signup and view all the answers

What is the main reason for using a mercury arc lamp as the light source in fluorescence microscopy?

It provides a high-intensity, wide wavelength range of light Signup and view all the answers

What is the term for the phenomenon where the peak of absorption and emission curves differ?

Stoke's shift Signup and view all the answers

What is the purpose of adding an anti-fade mounting medium to the slide in fluorescence microscopy?

To reduce photobleaching of the fluorophore Signup and view all the answers

What is the primary function of the barrier filter in a fluorescence microscope?

To filter out contaminating light from the arc lamp Signup and view all the answers

What is the term for the weakening or loss of fluorescence due to prolonged excitation?

Photobleaching Signup and view all the answers

What is the primary advantage of fluorescence microscopy over traditional light microscopy?

Ability to detect specific cell types or molecules Signup and view all the answers

What is the term for the component that combines the exciter filter, dichroic mirror, and barrier filter in a fluorescence microscope?

Filter cube Signup and view all the answers

What determines the excitation wavelength used in fluorescence microscopy?

The type of fluorophore used Signup and view all the answers

What is the primary purpose of the objective lens in a fluorescence microscope?

To focus the excitation light onto the sample Signup and view all the answers

What is the primary mechanism by which FISH exploits the ability of DNA strands to hybridize specifically?

Complementary sequences find each other and bind together Signup and view all the answers

What is the main advantage of using RNA probes in FISH?

All of the above Signup and view all the answers

What is the primary purpose of the dehydration step in FISH?

To facilitate hybridization of the probe and target DNA Signup and view all the answers

What is the main application of FISH in karyotyping and phylogenetic analysis?

To determine the evolutionary relationships between organisms Signup and view all the answers

What is the primary difference between FISH and FANA?

FISH uses fluorescent probes, while FANA uses radioactive probes Signup and view all the answers

What is the primary purpose of the Stoke's shift in fluorescence microscopy?

To emit light at a different wavelength Signup and view all the answers

What is the main advantage of using synthetic oligonucleotide probes in FISH?

All of the above Signup and view all the answers

What is the primary purpose of the xenon or mercury light source in fluorescence microscopy?

To provide a powerful light source for excitation Signup and view all the answers

What is the main advantage of using fluorescent labels in FISH?

They are more sensitive than radioactive labels Signup and view all the answers

What is the primary application of fluorescence microscopy in FISH?

To detect specific DNA sequences Signup and view all the answers

What is the primary purpose of using an ENA panel in autoimmune disease diagnosis?

To rule out other autoimmune disorders, such as Sjörgen syndrome, polymyositis, and scleroderma Signup and view all the answers

What is the 'gold standard' method for detecting ANA?

Indirect fluorescent antibody (IFA) Signup and view all the answers

What is the unit of measure reported in an immunoassay test for ANA?

U (arbitrary unit) Signup and view all the answers

What is the pattern of cellular fluorescence associated with SLE and mixed connective tissue disease?

Homogeneous (diffuse) Signup and view all the answers

What is the significance of a high titer in an IFA test for ANA?

The person is more likely to have an autoimmune disease Signup and view all the answers

What is the most common condition associated with a positive ANA test?

SLE Signup and view all the answers

What is the primary purpose of using additional laboratory tests, such as ESR and CRP, in autoimmune disease diagnosis?

To evaluate a person for inflammation Signup and view all the answers

What is the significance of a positive ANA test result in a person without symptoms?

It may take time to determine the significance of the result Signup and view all the answers

What is the purpose of reporting the pattern of cellular fluorescence in an IFA test for ANA?

To associate the ANA with a specific autoimmune disorder Signup and view all the answers

What is the significance of a negative ANA test result in a person with symptoms of an autoimmune disease?

The test is likely to be a false negative Signup and view all the answers

What percentage of people with Sjörgen syndrome have a positive ANA test result?

40-70% Signup and view all the answers

What is the primary application of flow cytometry in hematology?

Immunophenotyping of whole blood and bone marrow Signup and view all the answers

What is the purpose of the Kleihauer-Betke acid-elution test?

To detect fetal red cells in maternal blood Signup and view all the answers

What is the advantage of using flow cytometry over traditional methods?

It provides rapid analysis of multiple characteristics of single cells Signup and view all the answers

What percentage of people with scleroderma have a positive ANA test result?

60-90% Signup and view all the answers

What is the primary advantage of flow cytometry in the clinical laboratory?

It is more user-friendly and has a wider range of clinical applications Signup and view all the answers

What is the purpose of flow cytometry in immunophenotyping?

To analyze cell surface and cytoplasmic antigens Signup and view all the answers

What is the primary application of flow cytometry in the detection of fetal red cells in maternal blood?

To detect the presence of fetal red cells Signup and view all the answers

What is the advantage of using monoclonal antibodies in flow cytometry?

They are more specific than polyclonal antibodies Signup and view all the answers

What is the primary purpose of using Rh-immune globulin in pregnant women?

To prevent alloimmunization Signup and view all the answers

What is the primary advantage of using flow cytometry for the detection of fetal cells compared to the Kleihauer-Betke test?

More objective, reproducible, and sensitive results Signup and view all the answers

What is the primary advantage of using fluorescent dyes that bind to residual RNA in reticulocyte counting?

Excellent discrimination between reticulocytes and mature RBCs Signup and view all the answers

What is the primary importance of CD4 counts in HIV-infected individuals?

Assessing immune status Signup and view all the answers

What is the primary advantage of using flow cytometry in immunophenotyping of leukemias and lymphomas?

Rapid and effective diagnosis Signup and view all the answers

What is the primary application of flow cytometry in hematology?

All of the above Signup and view all the answers

What is the primary advantage of using antibodies to hemoglobin F in fetal cell detection?

More objective and sensitive results Signup and view all the answers

What is the primary importance of CD4 counts in HIV-infected individuals, compared to viral load testing?

CD4 counts directly assess immune status, while viral load testing measures viral load Signup and view all the answers

What is the primary advantage of using flow cytometry in distinguishing myeloid and lymphoid lineages in acute leukemias?

More effective distinction between lineages Signup and view all the answers

What is the primary application of flow cytometry in the diagnosis of leukemias and lymphomas?

All of the above Signup and view all the answers

What is the primary advantage of using flow cytometry in hematology, compared to traditional methods?

More objective, reproducible, and sensitive results Signup and view all the answers

What is the purpose of allowing the xenon or mercury light source to warm up for 15 minutes?

To allow the light source to reach constant illumination Signup and view all the answers

What is the function of the filter cube in fluorescence microscopy?

To select the correct wavelength for the fluorescent dye Signup and view all the answers

What is the purpose of using antibodies conjugated to fluorescent compounds in fluorescence microscopy?

To label specific proteins of interest Signup and view all the answers

What is the advantage of using fluorescence recovery after photobleaching (FRAP) over standard fluorescence microscopy?

It enables the study of protein dynamics and kinetics Signup and view all the answers

What is the primary application of fluorescent microscopy in the study of autoimmune disorders?

Imaging of proteins labeled with antibodies conjugated to fluorescent compounds Signup and view all the answers

What is the purpose of the antinuclear antibody (ANA) test?

To diagnose autoimmune disorders Signup and view all the answers

What is the difference between incident light and emitted light in fluorescence microscopy?

Incident light has a shorter wavelength, while emitted light has a longer wavelength Signup and view all the answers

What is the purpose of using fluorescently labeled macromolecular assemblies in fluorescence speckle microscopy?

To study the movement and turnover kinetics of cytoskeletal proteins Signup and view all the answers

What is the advantage of using fluorophores with different emission wavelengths in fluorescence microscopy?

It allows for the imaging of multiple structures or molecules simultaneously Signup and view all the answers

What is the purpose of keeping the exposure time constant when comparing features with the same dye on different samples?

To ensure consistent imaging results Signup and view all the answers

Study Notes

Analysis of DNA Mutations and Polymorphisms

Analysis of DNA mutations and polymorphisms involves various laboratory procedures that use electrophoresis to observe the sizes or amounts of nucleic acid.
Electrophoresis is the movement of particles under the force of an electric current, and it can be used to separate particles in gas, liquid, or solid phases.