5. GENE MUTATION SUMMARY

Question 1

Types of Mutations

Answer 1

Includes point mutations, single nucleotide polymorphisms (SNPs), deletions, insertions, and complex mutations (indels, duplications, inversions).

Question 2

Mutation Detection Techniques

Answer 2

Immunohistochemistry
High-Performance Liquid Chromatography
Gas Chromatography

Question 3

Used to visualize the presence and location of proteins within tissue samples. Common in detecting specific biomarkers associated with genetic mutations.

Answer 3

Immunohistochemistry

Question 4

Separates compounds based on their interactions with a stationary phase, allowing for precise analysis of small molecules, including mutation-related metabolites.

Answer 4

High-Performance Liquid Chromatography

Question 5

Similar to HPLC, but uses a gas phase for separation, particularly effective for volatile compounds. Useful in detecting metabolic changes due to gene mutations

Answer 5

Gas Chromatography

Question 6

Emphasizes techniques that enable detection, characterization, and categorization of mutations across diverse genetic materials

Answer 6

Mutation Screening Techniques

Question 7

Molecular Diagnostic Methods

Answer 7

Mass Spectrometry
Nucleic Acid Analysis Techniques

Question 8

Nucleic Acid Analysis Techniques

Answer 8

Hybridization-Based Methods
Melt-Curve Analysis
Heteroduplex Analysis
Array Technology

Question 9

Techniques like allele-specific oligomer hybridization target specific DNA sequences by using probes that bind only if sequences are fully complementary.

Answer 9

Hybridization-Based Methods

Question 10

Measures DNA melting temperatures to detect specific nucleotide variations, often used to identify SNPs or mutations in a fast, cost-effective manner.

Answer 10

Melt-Curve Analysis

Question 11

Detects mismatched DNA strands by creating heteroduplexes; mismatches alter the duplex structure, making it possible to identify mutations.

Answer 11

Heteroduplex Analysis

Question 12

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.

Answer 12

Array Technology

Question 13

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.

Answer 13

Mass Spectrometry

Question 14

Sequencing (Polymerization)-Based Methods

Answer 14

Sequence-Specific PCR
Allelic Discrimination with Fluorogenic Probes
Restriction Fragment Length Polymorphisms
Nonisotopic RNase Cleavage Assay
Cleavase Assay

Question 15

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.

Answer 15

Sequence-Specific PCR

Question 16

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.

Answer 16

Allelic Discrimination with Fluorogenic Probes

Question 17

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.

Answer 17

Restriction Fragment Length Polymorphisms

Question 18

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.

Answer 18

Nonisotopic RNase Cleavage Assay

Question 19

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.

Answer 19

Cleavase Assay

Question 20

Other Mutation Screening Methods

Answer 20

Development of High-Throughput Techniques
Combination Methods
Array-Based and Massive Parallel Sequencing
Cost Considerations
Method Selection Criteria

Question 21

Evolving needs in clinical laboratories have led to the creation of high-throughput and multiplex methods for comprehensive mutation screening.

Answer 21

Development of High-Throughput Techniques

Question 22

Integrating techniques, such as RFLP with modified primers, has improved the sensitivity and specificity of mutation detection

Answer 22

Combination Methods

Question 23

Provide high sensitivity and enable multiplex detection, essential for clinical diagnostics

Answer 23

Array-Based and Massive Parallel Sequencing

Question 24

The decreasing cost of instruments and reagents is making high-throughput screening more accessible, especially valuable for generating extensive genetic data per test

Answer 24

Cost Considerations

Question 25

Choice of method is determined by factors like instrumentation, target gene, mutation type, and the specific needs of the clinical application

Answer 25

Method Selection Criteria

Question 26

provides standardized nomenclature, continually updated, to ensure clarity in mutation reporting.

Answer 26

Human Genome Variation Society

Question 27

provides rules for gene naming to ensure consistency in genetic reporting

Answer 27

Human Genome Organization