5. GENE MUTATION SUMMARY Flashcards
(27 cards)
Types of Mutations
Includes point mutations, single nucleotide polymorphisms (SNPs), deletions, insertions, and complex mutations (indels, duplications, inversions).
Mutation Detection Techniques
Immunohistochemistry
High-Performance Liquid Chromatography
Gas Chromatography
Used to visualize the presence and location of proteins within tissue samples. Common in detecting specific biomarkers associated with genetic mutations.
Immunohistochemistry
Separates compounds based on their interactions with a stationary phase, allowing for precise analysis of small molecules, including mutation-related metabolites.
High-Performance Liquid Chromatography
Similar to HPLC, but uses a gas phase for separation, particularly effective for volatile compounds. Useful in detecting metabolic changes due to gene mutations
Gas Chromatography
Emphasizes techniques that enable detection, characterization, and categorization of mutations across diverse genetic materials
Mutation Screening Techniques
Molecular Diagnostic Methods
Mass Spectrometry
Nucleic Acid Analysis Techniques
Nucleic Acid Analysis Techniques
Hybridization-Based Methods
Melt-Curve Analysis
Heteroduplex Analysis
Array Technology
Techniques like allele-specific oligomer hybridization target specific DNA sequences by using probes that bind only if sequences are fully complementary.
Hybridization-Based Methods
Measures DNA melting temperatures to detect specific nucleotide variations, often used to identify SNPs or mutations in a fast, cost-effective manner.
Melt-Curve Analysis
Detects mismatched DNA strands by creating heteroduplexes; mismatches alter the duplex structure, making it possible to identify mutations.
Heteroduplex Analysis
High-throughput technology that allows simultaneous analysis of multiple genes. Arrays use probes to detect gene expression levels, SNPs, and mutations, supporting large-scale screening in clinical diagnostics.
Array Technology
Analytical technique that measures the mass-to-charge ratio of ions, highly useful for identifying and quantifying biomolecules. Applied in molecular diagnostics for analyzing proteins, nucleic acids, and complex biological samples.
Mass Spectrometry
Sequencing (Polymerization)-Based Methods
Sequence-Specific PCR
Allelic Discrimination with Fluorogenic Probes
Restriction Fragment Length Polymorphisms
Nonisotopic RNase Cleavage Assay
Cleavase Assay
Utilizes primers with specific 3′ ends that must match the target sequence, enabling detection of SNPs or mutations in PCR amplification.
Modifications like multiplexing (e.g., Amplification Refractory Mutation System (ARMS) PCR, tetra-primer PCR) enhance the capacity to detect multiple variations simultaneously, ideal for high-resolution genetic typing.
Sequence-Specific PCR
Real-time PCR technique employing fluorescent probes that distinguish between normal and mutant alleles based on color (e.g., FAM for one allele, VIC for the other).
Highly specific and sensitive, allowing precise allelic discrimination in real-time.
Allelic Discrimination with Fluorogenic Probes
Method to detect mutations by using restriction enzymes that cut DNA at specific sites, often altered by mutations.
Involves PCR amplification around the mutation site, followed by enzyme digestion and electrophoresis to detect pattern differences.
Restriction Fragment Length Polymorphisms
Relies on heteroduplex formation and RNase enzyme cleavage at mismatch sites.
Generates duplex RNA from PCR products with promoter sequences; mismatches are cleaved and analyzed via gel electrophoresis, providing a non-radioactive, efficient mutation screening.
Nonisotopic RNase Cleavage Assay
Utilizes the Cleavase enzyme to identify normal or mutant hybridized probes.
Produces a fluorescent signal upon detecting a mutation, offering high sensitivity for clinical mutation detection.
Cleavase Assay
Other Mutation Screening Methods
Development of High-Throughput Techniques
Combination Methods
Array-Based and Massive Parallel Sequencing
Cost Considerations
Method Selection Criteria
Evolving needs in clinical laboratories have led to the creation of high-throughput and multiplex methods for comprehensive mutation screening.
Development of High-Throughput Techniques
Integrating techniques, such as RFLP with modified primers, has improved the sensitivity and specificity of mutation detection
Combination Methods
Provide high sensitivity and enable multiplex detection, essential for clinical diagnostics
Array-Based and Massive Parallel Sequencing
The decreasing cost of instruments and reagents is making high-throughput screening more accessible, especially valuable for generating extensive genetic data per test
Cost Considerations
