5. GENE MUTATION Flashcards
What are types of gene mutations?
Deletions, insertions, inversions, translocations, and other changes affecting one base pair to thousands.
How can large differences in DNA sequence affect proteins?
They likely have significant effects on protein sequence
What is the effect range of point mutations on protein sequences?
They can cause various effects; changing one or a few base pairs may or may not alter the amino acid
Which sequencing method is used to analyze point mutations and their context?
Sequencing, which also provides the context of neighboring bases
What additional information can some sequencing methods provide?
The percentage of variant alleles relative to a reference sequence
What are limitations of different sequencing technologies?
Difficulty with detecting structural chromosomal abnormalities and copy-number variations
What is required to confirm germline variants detected by NGS in genetic testing?
Confirmation by Sanger sequencing or other methods
How are phenotypic changes in protein structure predicted?
By analyzing the nucleotide sequence
Why are standard biochemical, cytogenetic, and molecular methods still important?
They are used for analyzing frequently occurring variants via simple and inexpensive tests.
Why might DNA sequence changes not alter the amino acid sequence?
Due to the redundancy in codons for most amino acids.
What are the three outcomes of nucleotide substitutions?
Silent (no amino acid change), conservative (similar properties), or nonconservative (biochemically different amino acid).
A mutation that replaces an amino acid codon with a stop codon, causing premature protein termination
nonsense mutation
What percentage of disease-related gene lesions are nonsense mutations?
About 11%
What results from insertion or deletion of nucleotides not in multiples of three?
A frameshift mutation, altering the triplet reading frame
Why do frameshifts often lead to premature stop codons?
The genetic code structure often brings a stop codon sooner in the out-of-frame sequence
Where do nonconservative, nonsense, and frameshift mutations cause different phenotypes
Depending on their location along the protein sequence
How does the mutation location affect the mutation’s impact?
Mutations at the 3’ end of coding regions often have minor effects, while those at the 5’ end may cause major alterations
Why might finding a change in DNA sequence not always indicate an altered phenotype?
Point mutations must be screened to identify silent, conservative, or nonconservative effects.
are specific DNA changes often inherited with disease phenotypes and are mapped near disease genes
single-nucleotide polymorphisms (SNPs)
Give an example of a gene with multiple disease-associated mutations
The cystic fibrosis transmembrane regulator (CFTR) gene has over 600 such mutations.
What challenge exists in detecting mutations in large genes
Screening across thousands of base pairs to identify a single altered nucleotide
What methods analyze the actual protein or amino acid alteration
Biochemical methods detect direct protein changes, especially in metabolic defects
How do immunoassays work in detecting various analytes?
They use antibodies to capture and detect target molecules in biological fluids
What is the principle of enzyme-linked immunosorbent assays (ELISAs)?
Use of capture and detection antibodies with a substrate to produce signals like color or light
What innovation reduced the radioactive hazards of radioimmunoassays (RIAs)?
Coupling enzymes like alkaline phosphatase with antibodies to generate a detectable signal.
Who first used ELISA to detect immunoglobulin G in rabbit serum, and in what year?
Engvall and Perlmann in 1971
What mutation effect does the genetic code structure likely help to prevent?
The formation of long nonsense proteins by frameshift mutations.
Who coined the term “antibody” and when?
Paul Ehrlich, 1891
Who described immunofluorescence staining on frozen sections and when?
Coons, in 1940.
When was immunohistochemistry (IHC) first performed on fixed tissues, and by whom?
By Taylor and Burns, 34 years after 1940
What improvement did monoclonal antibodies bring to IHC?
Less nonspecific staining and better image quality
How are monoclonal antibodies (mAbs) produced?
By fusing a single antibody-producing cell with an immortal cell, creating hybridomas.
What technique did Köhler and Milstein develop in 1975?
The hybridoma technique for producing monoclonal antibodies
The hybridoma technique for producing monoclonal antibodies is developed by and when
Köhler and Milstein develop in 1975?
What is the thickness of tissue slices for IHC on fixed and frozen sections?
Fixed: <5 microns; Frozen: 5-15 microns
Treating fixed tissue to uncover epitopes using enzyme digestion or heating
antigen retrieval
Why might snap frozen tissue be used in IHC?
To avoid epitope alteration by formalin fixation.
What is used to freeze tissue quickly for cryostat sectioning?
Isopentane, at −160°C.
What solutions minimize nonspecific binding in IHC?
Blocking solutions containing serum protein, detergent, or unlabeled antibodies
How are endogenous substances blocked to prevent background staining in IHC?
Using hydrogen peroxide, UV light, or 1% serum.
How are fluorescent signals generated in IHC?
By attaching fluor molecules to antibodies that emit signals upon excitation
How are colorimetric signals generated in IHC?
Through oxidation of a substrate by enzymes like horseradish peroxidase or alkaline phosphatase.
What is the primary advantage of indirect antibody staining over direct staining in IHC?
Greater sensitivity due to signal amplification.
What is the blocking, staining, and washing procedure used for in dual staining IHC?
To apply a second antibody for multi-color staining.
Why has IHC become increasingly important in pathology?
It helps assess tissue expression of target molecules for targeted therapies.
Who named chromatography, and from which language is it derived?
Mikhail S. Tswett, derived from Greek (chroma = color, graph = writing)
What are the two phases in High-Performance Liquid Chromatography (HPLC)?
Mobile phase (solvent) and stationary phase (solid support).
What type of chromatography column excludes larger molecules and elutes them faster?
Size-exclusion columns.
How does reverse-phase chromatography differ from normal-phase?
Lipophilic molecules elute slower in reverse-phase
What is affinity chromatography designed to do?
Immobilize specific ions while washing through others.
What types of detectors are used in HPLC?
Light scattering, fluorescence, refractive index, UV light absorption, and MS
What is the main improvement of ultra-HPLC (UHPLC) over HPLC?
Increased resolution and faster separation with less solvent.
What mobile phase does Gas Chromatography (GC) use?
An inert gas.
What are typical applications of GC, especially when coupled with MS?
Detection of drugs, poisons, and disease biomarkers in biological samples
What is the primary function of a mass spectrometer
To convert molecules to ions that can be moved in a magnetic field based on their charge and mass
How are ions generated in mass spectrometry?
High-energy electrons from an ion source hit target sample molecules, separating them into ions, usually cations
What happens to ions after they are generated in mass spectrometry?
The ions may fragment into smaller ions and neutral particles before being accelerated and focused into a beam.
How are ions directed to the detector in a mass spectrometer?
Ions are deflected according to their mass and charge by varying the strength of a magnetic field
What is displayed on the output spectrum of a mass spectrometer?
The mass/charge value is on the x-axis, and the abundance of the ion is on the y-axis.
What are the two primary ionization methods used for mass spectrometry of large biomolecules?
Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
How does electrospray ionization (ESI) work?
A test sample is converted into a fine spray of charged droplets that are electrostatically directed to the mass spectrometer inlet.
How are ions formed using MALDI?
A laser pulse is fired into a sample coated with a matrix, causing desorption and ionization of the sample molecules
What is the time of flight (TOF) principle in mass spectrometry?
Ions fly to the detector at a speed based on their mass and charge, allowing separation according to mass/charge ratio.
What is surface-enhanced laser desorption/ionization (SELDI)?
An extension of MALDI that combines time of flight for flexible identification and quantification of peptides
How can mass spectrometry be used to analyze nucleic acids?
By detecting single-base-pair changes using a primer extension technique that modifies the mass and charge of the primer.
What role does polymerase chain reaction (PCR) play in nucleic acid analysis?
PCR amplifies inherited mutations from convenient specimens like blood or buccal cells.
What challenges are associated with detecting somatic mutations?
Somatic mutations may be present in only a small fraction of the total specimen, making them difficult to detect.
What types of mutation detection methods exist?
Hybridization-based methods, sequence (polymerization)-based methods, and enzymatic or chemical cleavage methods
A method based on DNA’s preference for a double-stranded state, allowing the formation of three-dimensional conformers that can be analyzed
Single-Strand Conformation Polymorphism (SSCP)
How are single-stranded nucleic acids denatured for SSCP
By heating them in sodium hydroxide and formamide, then rapidly cooling
What is the detection method used for band or peak patterns in SSCP?
Silver stain, radioactivity, or fluorescence.
What percentage of putative mutations can SSCP detect
SSCP can detect 35% to 100% of putative mutations
What is required for reliable results in detecting somatic mutations using SSCP
A cell suspension that is at least 30% tumor cells or microdissection of solid tumor tissue.
What modifications of SSCP have been developed for increased sensitivity
RNA-SSCP (using RNA) and REF-SSCP (restriction endonuclease fingerprinting), though they are more difficult to interpret
A method that utilizes differences in melting temperatures of short DNA sequences (about 20 bases) with one or two mismatches compared to perfectly complementary sequences, using synthetic single-stranded probes for normal or mutant target DNA.
allele-specific oligomer hybridization (ASO)
What is the principle behind ASO hybridization?
At specific annealing temperatures and conditions, probes with perfect complementary sequences bind to target DNA, while those with mismatches do not.
What is the typical application of ASO analysis?
It is used to test for known mutations, such as BRCA1 and BRCA2 mutations in breast cancer and p16 mutations in familial melanoma
How is ASO analysis performed in a laboratory setting?
ASO can be done using a dot blot method with immobilized probes or in a reverse dot blot format using a 96-well plate, where amplifications are performed with PCR and detected through chromogenic or chemiluminescent substrates.
What does melt-curve analysis (MCA) measure?
MCA exploits the denaturation characteristics of DNA duplexes and is a post-amplification step of real-time PCR, analyzing DNA amplicons’ melting temperatures as the temperature increases
What fluorescent dyes are commonly used in MCA?
Ethidium bromide,
SYBR green, and
LC green a
re used as DNA-specific fluorescent dyes during PCR amplification
How does MCA differentiate sequences?
Specimens with identical sequences yield overlapping peaks at their melting temperature (Tm), while different sequences show separate peaks at different temperatures
What enhances the specificity of MCA?
High-resolution melt-curve analysis (HR-MCA) uses fluorescent resonance energy transfer (FRET) probes, which fluoresce only when bound to the target sequence.
A method that reveals mutations by hybridizing non-identical double-stranded DNA fragments, formed by heating and cooling, and analyzing their migration through gels.
heteroduplex analysis
How are heteroduplexes resolved in laboratory analysis?
Heteroduplexes are detected by their different migration patterns compared to homoduplexes in polyacrylamide or agarose gels.
What is the role of denaturing high-performance liquid chromatography (DHPLC) in heteroduplex analysis?
DHPLC separates PCR products, allowing for the detection of heteroduplexes based on their different elution times compared to homoduplexes.
achieves single-base-pair resolution through high-density oligonucleotide arrays, allowing simultaneous testing for numerous potential sequence mutations or SNPs.
Array technology
What is the process of analyzing DNA using array technology?
Test DNA is fragmented and bound to complementary probes on the array, with specific formats like standard and redundant tiling used for mutation detection
It tests multiple loci simultaneously using small samples, where bead color and test label indicate the presence of mutations or polymorphisms, and fluorescence is detected through flow cytometry
bead array
What is the primary purpose of sequence-specific (primer) PCR (SSP-PCR)?
To detect point mutations and other SNPs.
In SSP-PCR, where must the primer’s 3’ end match the template?
The nucleotide to be analyzed.
How are primers designed in SSP-PCR?
Primers are designed such that the 3’ end falls on the nucleotide to be analyzed, requiring perfect matching with the template for Taq polymerase extension.
What is the significance of the 3’ end of a primer in SSP-PCR?
The 3’ end must match the template perfectly to allow extension by Taq polymerase, indicating the presence or absence of a mutation.
What modifications can be made to SSP-PCR to distinguish normal and mutant sequences?
Modifications include varying the length of the normal or mutant primer to yield differently sized products, or multiplexing primers for simultaneous analysis
What is the application of SSP-PCR in clinical practice?
SSP-PCR is routinely used for high-resolution HLA typing and detecting commonly occurring mutations.
Describe the process of allelic discrimination with fluorogenic probes
This method uses two probes labeled with different fluorophores, one for the normal sequence and one for the mutant. The presence of a fluorescent signal indicates whether the test sequence is normal or mutant.
How does the probe hybridization affect fluorescence in allelic discrimination?
If the probe matches the test sequence, it is digested by polymerase, releasing the reporter dye. High fluorescence from one dye indicates a specific sequence, while low fluorescence from the other indicates the absence of that sequence.
is used to detect sequence alterations by amplifying the region surrounding a mutation and cutting the amplicon with the appropriate restriction enzyme.
Restriction Fragment Length Polymorphism (RFLP) analysis
How can RFLP analysis be affected by mutations
Mutations may inactivate a restriction site or create a new one, allowing for detection of mutations through changes in fragment sizes after enzyme digestion.
What are some examples of mutations detected by PCR-RFLP?
Commonly occurring mutations such as FLT3 kinase domain and HFE mutations can be detected using PCR-RFLP
is a heteroduplex analysis using duplex RNA, where amplifications are made with primers tailed with RNA polymerase promoters.
Nonisotopic RNase Cleavage Assay (NIRCA)?
How does NIRCA identify mutations?
It forms heteroduplexes between normal and mutant transcripts, and mismatches are cleaved by specific RNase enzymes, with remaining fragments analyzed by gel electrophoresis
uses a proprietary enzyme system to detect mutations by recognizing hybridized probe structures, producing a fluorescent signal upon cleavage.
Cleavase assay
What are the advantages of the Cleavase assay
Advantages include a short hands-on time and optional PCR amplification, applicable in various clinical molecular diagnostics areas.
What drives the discovery of new techniques and modifications in clinical laboratory methods for mutation screening?
Clinical laboratory requirements for robust, accurate, and sensitive assays.
What was a commonly used mutation screening method in clinical laboratories that revealed limitations leading to the development of diverse methods
Single-Strand Conformation Polymorphism (SSCP).
What strategies have been proposed to increase sensitivity and detection in mutation screening?
Combinations of methods, such as RFLP with modified primers.
What type of methods have become a main focus in molecular diagnostics for mutation detection?
Array-based methods and massive parallel sequencing methods
How have costs related to instrumentation and reagents for high-throughput methods changed over time?
Costs are decreasing, especially relative to the information generated per test.
What factors influence the performance of mutation detection methods?
Specimen type, template sequence, and type of mutation.
What is essential for accurate testing and reporting of gene mutations?
A descriptive and consistent system of expressing mutations and polymorphisms
How is the first nucleotide of the first amino acid in a sequence designated in gene variant nomenclature
Position + 1 (with preceding nucleotides as -1)
How is a substitution of a nucleotide expressed in gene variant nomenclature
By position or nucleotide interval, type of change, changed nucleotide, symbol “>”, and new nucleotide (e.g., c.7C>T).
How is an insertion of nucleotides denoted in gene variant nomenclature?
As c.position1_position2ins(inserted nucleotides)
How are gene mutations in recessive diseases indicated in nomenclature?
By designating each mutation separated by + (e.g., c.[2357C>T]; [2378delA])
What prefixes are recommended to distinguish between different types of sequences in mutation nomenclature?
“g.” for genomic DNA, “c.” for coding DNA, “m.” for mitochondrial DNA, “r.” for RNA, and “p.” for protein sequences
How are complex changes and multiple concurrent mutations reported?
They are reported as they occur, with the assumption that the 3′-most repeat is the one affected.
What guidelines does the Human Genome Organization (HUGO) provide for reporting gene names?
Gene names should be capitalized and italicized without hyphens; protein names are not italicized or fully capitalized
How is the KRAS gene and its corresponding protein named in gene nomenclature?
The KRAS gene codes for the K-Ras protein; the gene name is italicized (KRAS), and the protein name is not italicized (K-Ras).
