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MOLECULAR BIOLOGY > 5. GENE MUTATION > Flashcards

5. GENE MUTATION Flashcards

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1
Q

What are types of gene mutations?

A

Deletions, insertions, inversions, translocations, and other changes affecting one base pair to thousands.

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2
Q

How can large differences in DNA sequence affect proteins?

A

They likely have significant effects on protein sequence

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3
Q

What is the effect range of point mutations on protein sequences?

A

They can cause various effects; changing one or a few base pairs may or may not alter the amino acid

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4
Q

Which sequencing method is used to analyze point mutations and their context?

A

Sequencing, which also provides the context of neighboring bases

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5
Q

What additional information can some sequencing methods provide?

A

The percentage of variant alleles relative to a reference sequence

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6
Q

What are limitations of different sequencing technologies?

A

Difficulty with detecting structural chromosomal abnormalities and copy-number variations

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7
Q

What is required to confirm germline variants detected by NGS in genetic testing?

A

Confirmation by Sanger sequencing or other methods

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8
Q

How are phenotypic changes in protein structure predicted?

A

By analyzing the nucleotide sequence

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9
Q

Why are standard biochemical, cytogenetic, and molecular methods still important?

A

They are used for analyzing frequently occurring variants via simple and inexpensive tests.

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10
Q

Why might DNA sequence changes not alter the amino acid sequence?

A

Due to the redundancy in codons for most amino acids.

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11
Q

What are the three outcomes of nucleotide substitutions?

A

Silent (no amino acid change), conservative (similar properties), or nonconservative (biochemically different amino acid).

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12
Q

A mutation that replaces an amino acid codon with a stop codon, causing premature protein termination

A

nonsense mutation

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13
Q

What percentage of disease-related gene lesions are nonsense mutations?

A

About 11%

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14
Q

What results from insertion or deletion of nucleotides not in multiples of three?

A

A frameshift mutation, altering the triplet reading frame

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15
Q

Why do frameshifts often lead to premature stop codons?

A

The genetic code structure often brings a stop codon sooner in the out-of-frame sequence

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16
Q

Where do nonconservative, nonsense, and frameshift mutations cause different phenotypes

A

Depending on their location along the protein sequence

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17
Q

How does the mutation location affect the mutation’s impact?

A

Mutations at the 3’ end of coding regions often have minor effects, while those at the 5’ end may cause major alterations

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18
Q

Why might finding a change in DNA sequence not always indicate an altered phenotype?

A

Point mutations must be screened to identify silent, conservative, or nonconservative effects.

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19
Q

are specific DNA changes often inherited with disease phenotypes and are mapped near disease genes

A

single-nucleotide polymorphisms (SNPs)

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20
Q

Give an example of a gene with multiple disease-associated mutations

A

The cystic fibrosis transmembrane regulator (CFTR) gene has over 600 such mutations.

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21
Q

What challenge exists in detecting mutations in large genes

A

Screening across thousands of base pairs to identify a single altered nucleotide

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22
Q

What methods analyze the actual protein or amino acid alteration

A

Biochemical methods detect direct protein changes, especially in metabolic defects

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23
Q

How do immunoassays work in detecting various analytes?

A

They use antibodies to capture and detect target molecules in biological fluids

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24
Q

What is the principle of enzyme-linked immunosorbent assays (ELISAs)?

A

Use of capture and detection antibodies with a substrate to produce signals like color or light

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25
Q

What innovation reduced the radioactive hazards of radioimmunoassays (RIAs)?

A

Coupling enzymes like alkaline phosphatase with antibodies to generate a detectable signal.

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26
Q

Who first used ELISA to detect immunoglobulin G in rabbit serum, and in what year?

A

Engvall and Perlmann in 1971

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27
Q

What mutation effect does the genetic code structure likely help to prevent?

A

The formation of long nonsense proteins by frameshift mutations.

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28
Q

Who coined the term “antibody” and when?

A

Paul Ehrlich, 1891

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29
Q

Who described immunofluorescence staining on frozen sections and when?

A

Coons, in 1940.

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30
Q

When was immunohistochemistry (IHC) first performed on fixed tissues, and by whom?

A

By Taylor and Burns, 34 years after 1940

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31
Q

What improvement did monoclonal antibodies bring to IHC?

A

Less nonspecific staining and better image quality

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32
Q

How are monoclonal antibodies (mAbs) produced?

A

By fusing a single antibody-producing cell with an immortal cell, creating hybridomas.

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33
Q

What technique did Köhler and Milstein develop in 1975?

A

The hybridoma technique for producing monoclonal antibodies

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34
Q

The hybridoma technique for producing monoclonal antibodies is developed by and when

A

Köhler and Milstein develop in 1975?

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35
Q

What is the thickness of tissue slices for IHC on fixed and frozen sections?

A

Fixed: <5 microns; Frozen: 5-15 microns

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36
Q

Treating fixed tissue to uncover epitopes using enzyme digestion or heating

A

antigen retrieval

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37
Q

Why might snap frozen tissue be used in IHC?

A

To avoid epitope alteration by formalin fixation.

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38
Q

What is used to freeze tissue quickly for cryostat sectioning?

A

Isopentane, at −160°C.

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39
Q

What solutions minimize nonspecific binding in IHC?

A

Blocking solutions containing serum protein, detergent, or unlabeled antibodies

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40
Q

How are endogenous substances blocked to prevent background staining in IHC?

A

Using hydrogen peroxide, UV light, or 1% serum.

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41
Q

How are fluorescent signals generated in IHC?

A

By attaching fluor molecules to antibodies that emit signals upon excitation

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42
Q

How are colorimetric signals generated in IHC?

A

Through oxidation of a substrate by enzymes like horseradish peroxidase or alkaline phosphatase.

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43
Q

What is the primary advantage of indirect antibody staining over direct staining in IHC?

A

Greater sensitivity due to signal amplification.

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44
Q

What is the blocking, staining, and washing procedure used for in dual staining IHC?

A

To apply a second antibody for multi-color staining.

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45
Q

Why has IHC become increasingly important in pathology?

A

It helps assess tissue expression of target molecules for targeted therapies.

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46
Q

Who named chromatography, and from which language is it derived?

A

Mikhail S. Tswett, derived from Greek (chroma = color, graph = writing)

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47
Q

What are the two phases in High-Performance Liquid Chromatography (HPLC)?

A

Mobile phase (solvent) and stationary phase (solid support).

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48
Q

What type of chromatography column excludes larger molecules and elutes them faster?

A

Size-exclusion columns.

49
Q

How does reverse-phase chromatography differ from normal-phase?

A

Lipophilic molecules elute slower in reverse-phase

50
Q

What is affinity chromatography designed to do?

A

Immobilize specific ions while washing through others.

51
Q

What types of detectors are used in HPLC?

A

Light scattering, fluorescence, refractive index, UV light absorption, and MS

52
Q

What is the main improvement of ultra-HPLC (UHPLC) over HPLC?

A

Increased resolution and faster separation with less solvent.

53
Q

What mobile phase does Gas Chromatography (GC) use?

A

An inert gas.

54
Q

What are typical applications of GC, especially when coupled with MS?

A

Detection of drugs, poisons, and disease biomarkers in biological samples

55
Q

What is the primary function of a mass spectrometer

A

To convert molecules to ions that can be moved in a magnetic field based on their charge and mass

56
Q

How are ions generated in mass spectrometry?

A

High-energy electrons from an ion source hit target sample molecules, separating them into ions, usually cations

57
Q

What happens to ions after they are generated in mass spectrometry?

A

The ions may fragment into smaller ions and neutral particles before being accelerated and focused into a beam.

58
Q

How are ions directed to the detector in a mass spectrometer?

A

Ions are deflected according to their mass and charge by varying the strength of a magnetic field

59
Q

What is displayed on the output spectrum of a mass spectrometer?

A

The mass/charge value is on the x-axis, and the abundance of the ion is on the y-axis.

60
Q

What are the two primary ionization methods used for mass spectrometry of large biomolecules?

A

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).

61
Q

How does electrospray ionization (ESI) work?

A

A test sample is converted into a fine spray of charged droplets that are electrostatically directed to the mass spectrometer inlet.

62
Q

How are ions formed using MALDI?

A

A laser pulse is fired into a sample coated with a matrix, causing desorption and ionization of the sample molecules

63
Q

What is the time of flight (TOF) principle in mass spectrometry?

A

Ions fly to the detector at a speed based on their mass and charge, allowing separation according to mass/charge ratio.

64
Q

What is surface-enhanced laser desorption/ionization (SELDI)?

A

An extension of MALDI that combines time of flight for flexible identification and quantification of peptides

65
Q

How can mass spectrometry be used to analyze nucleic acids?

A

By detecting single-base-pair changes using a primer extension technique that modifies the mass and charge of the primer.

66
Q

What role does polymerase chain reaction (PCR) play in nucleic acid analysis?

A

PCR amplifies inherited mutations from convenient specimens like blood or buccal cells.

67
Q

What challenges are associated with detecting somatic mutations?

A

Somatic mutations may be present in only a small fraction of the total specimen, making them difficult to detect.

68
Q

What types of mutation detection methods exist?

A

Hybridization-based methods, sequence (polymerization)-based methods, and enzymatic or chemical cleavage methods

69
Q

A method based on DNA’s preference for a double-stranded state, allowing the formation of three-dimensional conformers that can be analyzed

A

Single-Strand Conformation Polymorphism (SSCP)

70
Q

How are single-stranded nucleic acids denatured for SSCP

A

By heating them in sodium hydroxide and formamide, then rapidly cooling

71
Q

What is the detection method used for band or peak patterns in SSCP?

A

Silver stain, radioactivity, or fluorescence.

72
Q

What percentage of putative mutations can SSCP detect

A

SSCP can detect 35% to 100% of putative mutations

73
Q

What is required for reliable results in detecting somatic mutations using SSCP

A

A cell suspension that is at least 30% tumor cells or microdissection of solid tumor tissue.

74
Q

What modifications of SSCP have been developed for increased sensitivity

A

RNA-SSCP (using RNA) and REF-SSCP (restriction endonuclease fingerprinting), though they are more difficult to interpret

75
Q

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.

A

allele-specific oligomer hybridization (ASO)

76
Q

What is the principle behind ASO hybridization?

A

At specific annealing temperatures and conditions, probes with perfect complementary sequences bind to target DNA, while those with mismatches do not.

77
Q

What is the typical application of ASO analysis?

A

It is used to test for known mutations, such as BRCA1 and BRCA2 mutations in breast cancer and p16 mutations in familial melanoma

78
Q

How is ASO analysis performed in a laboratory setting?

A

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.

79
Q

What does melt-curve analysis (MCA) measure?

A

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

80
Q

What fluorescent dyes are commonly used in MCA?

A

Ethidium bromide,
SYBR green, and
LC green a

re used as DNA-specific fluorescent dyes during PCR amplification

81
Q

How does MCA differentiate sequences?

A

Specimens with identical sequences yield overlapping peaks at their melting temperature (Tm), while different sequences show separate peaks at different temperatures

82
Q

What enhances the specificity of MCA?

A

High-resolution melt-curve analysis (HR-MCA) uses fluorescent resonance energy transfer (FRET) probes, which fluoresce only when bound to the target sequence.

83
Q

A method that reveals mutations by hybridizing non-identical double-stranded DNA fragments, formed by heating and cooling, and analyzing their migration through gels.

A

heteroduplex analysis

84
Q

How are heteroduplexes resolved in laboratory analysis?

A

Heteroduplexes are detected by their different migration patterns compared to homoduplexes in polyacrylamide or agarose gels.

85
Q

What is the role of denaturing high-performance liquid chromatography (DHPLC) in heteroduplex analysis?

A

DHPLC separates PCR products, allowing for the detection of heteroduplexes based on their different elution times compared to homoduplexes.

86
Q

achieves single-base-pair resolution through high-density oligonucleotide arrays, allowing simultaneous testing for numerous potential sequence mutations or SNPs.

A

Array technology

87
Q

What is the process of analyzing DNA using array technology?

A

Test DNA is fragmented and bound to complementary probes on the array, with specific formats like standard and redundant tiling used for mutation detection

88
Q

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

A

bead array

89
Q

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

A

To detect point mutations and other SNPs.

90
Q

In SSP-PCR, where must the primer’s 3’ end match the template?

A

The nucleotide to be analyzed.

91
Q

How are primers designed in SSP-PCR?

A

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.

92
Q

What is the significance of the 3’ end of a primer in SSP-PCR?

A

The 3’ end must match the template perfectly to allow extension by Taq polymerase, indicating the presence or absence of a mutation.

93
Q

What modifications can be made to SSP-PCR to distinguish normal and mutant sequences?

A

Modifications include varying the length of the normal or mutant primer to yield differently sized products, or multiplexing primers for simultaneous analysis

94
Q

What is the application of SSP-PCR in clinical practice?

A

SSP-PCR is routinely used for high-resolution HLA typing and detecting commonly occurring mutations.

95
Q

Describe the process of allelic discrimination with fluorogenic probes

A

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.

96
Q

How does the probe hybridization affect fluorescence in allelic discrimination?

A

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.

97
Q

is used to detect sequence alterations by amplifying the region surrounding a mutation and cutting the amplicon with the appropriate restriction enzyme.

A

Restriction Fragment Length Polymorphism (RFLP) analysis

98
Q

How can RFLP analysis be affected by mutations

A

Mutations may inactivate a restriction site or create a new one, allowing for detection of mutations through changes in fragment sizes after enzyme digestion.

99
Q

What are some examples of mutations detected by PCR-RFLP?

A

Commonly occurring mutations such as FLT3 kinase domain and HFE mutations can be detected using PCR-RFLP

100
Q

is a heteroduplex analysis using duplex RNA, where amplifications are made with primers tailed with RNA polymerase promoters.

A

Nonisotopic RNase Cleavage Assay (NIRCA)?

101
Q

How does NIRCA identify mutations?

A

It forms heteroduplexes between normal and mutant transcripts, and mismatches are cleaved by specific RNase enzymes, with remaining fragments analyzed by gel electrophoresis

102
Q

uses a proprietary enzyme system to detect mutations by recognizing hybridized probe structures, producing a fluorescent signal upon cleavage.

A

Cleavase assay

103
Q

What are the advantages of the Cleavase assay

A

Advantages include a short hands-on time and optional PCR amplification, applicable in various clinical molecular diagnostics areas.

104
Q

What drives the discovery of new techniques and modifications in clinical laboratory methods for mutation screening?

A

Clinical laboratory requirements for robust, accurate, and sensitive assays.

105
Q

What was a commonly used mutation screening method in clinical laboratories that revealed limitations leading to the development of diverse methods

A

Single-Strand Conformation Polymorphism (SSCP).

106
Q

What strategies have been proposed to increase sensitivity and detection in mutation screening?

A

Combinations of methods, such as RFLP with modified primers.

107
Q

What type of methods have become a main focus in molecular diagnostics for mutation detection?

A

Array-based methods and massive parallel sequencing methods

108
Q

How have costs related to instrumentation and reagents for high-throughput methods changed over time?

A

Costs are decreasing, especially relative to the information generated per test.

109
Q

What factors influence the performance of mutation detection methods?

A

Specimen type, template sequence, and type of mutation.

110
Q

What is essential for accurate testing and reporting of gene mutations?

A

A descriptive and consistent system of expressing mutations and polymorphisms

111
Q

How is the first nucleotide of the first amino acid in a sequence designated in gene variant nomenclature

A

Position + 1 (with preceding nucleotides as -1)

112
Q

How is a substitution of a nucleotide expressed in gene variant nomenclature

A

By position or nucleotide interval, type of change, changed nucleotide, symbol “>”, and new nucleotide (e.g., c.7C>T).

113
Q

How is an insertion of nucleotides denoted in gene variant nomenclature?

A

As c.position1_position2ins(inserted nucleotides)

114
Q

How are gene mutations in recessive diseases indicated in nomenclature?

A

By designating each mutation separated by + (e.g., c.[2357C>T]; [2378delA])

115
Q

What prefixes are recommended to distinguish between different types of sequences in mutation nomenclature?

A

“g.” for genomic DNA, “c.” for coding DNA, “m.” for mitochondrial DNA, “r.” for RNA, and “p.” for protein sequences

116
Q

How are complex changes and multiple concurrent mutations reported?

A

They are reported as they occur, with the assumption that the 3′-most repeat is the one affected.

117
Q

What guidelines does the Human Genome Organization (HUGO) provide for reporting gene names?

A

Gene names should be capitalized and italicized without hyphens; protein names are not italicized or fully capitalized

118
Q

How is the KRAS gene and its corresponding protein named in gene nomenclature?

A

The KRAS gene codes for the K-Ras protein; the gene name is italicized (KRAS), and the protein name is not italicized (K-Ras).

MOLECULAR BIOLOGY (17 decks)

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