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Questions and Answers
What are the two main types of material that can be used in genetic testing?
What are the two main types of material that can be used in genetic testing?
What is the best general source of DNA for genetic testing?
What is the best general source of DNA for genetic testing?
Peripheral blood
Which of the following DNA sources is technically very demanding?
Which of the following DNA sources is technically very demanding?
Amniotic fluid is considered a better source of fetal DNA compared to chorionic villi.
Amniotic fluid is considered a better source of fetal DNA compared to chorionic villi.
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Which of the following techniques can be used as a promising alternative to chorionic villi sampling for prenatal diagnosis?
Which of the following techniques can be used as a promising alternative to chorionic villi sampling for prenatal diagnosis?
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The first step in genetic testing almost always involves amplification of the DNA or RNA sample using PCR.
The first step in genetic testing almost always involves amplification of the DNA or RNA sample using PCR.
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What is the primary method used for mutation scanning during genetic testing?
What is the primary method used for mutation scanning during genetic testing?
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When does DNA sequencing become problematic?
When does DNA sequencing become problematic?
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What are the two types of sequencing techniques discussed in the text?
What are the two types of sequencing techniques discussed in the text?
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Next Generation Sequencing (NGS) is considered massively parallel, meaning it can sequence millions of fragments simultaneously.
Next Generation Sequencing (NGS) is considered massively parallel, meaning it can sequence millions of fragments simultaneously.
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The critical difference between Sanger Sequencing and Next Generation Sequencing is the sequencing volume.
The critical difference between Sanger Sequencing and Next Generation Sequencing is the sequencing volume.
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What is the gold standard for mutation detection?
What is the gold standard for mutation detection?
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What are some of the common methods used for scanning a gene for mutations before sequencing?
What are some of the common methods used for scanning a gene for mutations before sequencing?
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Most mutations are found in heterozygous form, even in autosomal recessive conditions.
Most mutations are found in heterozygous form, even in autosomal recessive conditions.
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Heteroduplexes can be formed simply by heating a PCR product to denature it, and then cooling it slowly.
Heteroduplexes can be formed simply by heating a PCR product to denature it, and then cooling it slowly.
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The protein truncation test (PTT) is a specific test for mutations that create premature termination codons, such as frameshifts or splice site mutations.
The protein truncation test (PTT) is a specific test for mutations that create premature termination codons, such as frameshifts or splice site mutations.
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What are some of the advantages of using microarrays for mutation detection?
What are some of the advantages of using microarrays for mutation detection?
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Genomewide studies of methylation patterns typically involve chromatin immunoprecipitation using an antibody against methylated DNA.
Genomewide studies of methylation patterns typically involve chromatin immunoprecipitation using an antibody against methylated DNA.
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What are the two main methods used to study the methylation status of an individual sequence?
What are the two main methods used to study the methylation status of an individual sequence?
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The restriction enzyme MspI can only cut unmethylated CCGG sequences, while Hpall can cut both methylated and unmethylated CCGG sequences.
The restriction enzyme MspI can only cut unmethylated CCGG sequences, while Hpall can cut both methylated and unmethylated CCGG sequences.
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What is the primary issue associated with unclassified genetic variants?
What is the primary issue associated with unclassified genetic variants?
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What are some of the suggestions for classifying unclassified genetic variants?
What are some of the suggestions for classifying unclassified genetic variants?
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Testing for the presence or absence of a known sequence change is more straightforward than scanning a gene for any mutation.
Testing for the presence or absence of a known sequence change is more straightforward than scanning a gene for any mutation.
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What are some of the methods used for testing a specified mutation or SNP?
What are some of the methods used for testing a specified mutation or SNP?
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In certain instances, restriction sites can be engineered into PCR products to facilitate testing for specific mutations.
In certain instances, restriction sites can be engineered into PCR products to facilitate testing for specific mutations.
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What are some typical applications that involve testing for a specified sequence change?
What are some typical applications that involve testing for a specified sequence change?
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Study Notes
Human Molecular Genetics - Chapter 18: Genetic Testing of Individuals
- Genetic testing is unique among clinical tests, generally performed only once, and forming a permanent part of a patient's health record. Accuracy is critically important to avoid errors.
Key Concepts
- Genetic testing can utilize either DNA or RNA.
- RNA testing requires tissue where the gene of interest is expressed, and necessitates more careful handling than DNA samples.
- RNA analysis may be more cost-effective for genes with many small exons, and can reveal abnormal splicing not apparent in DNA testing.
- Amplifying relevant sequences (using PCR or RT-PCR for RNA) is often the first step in testing.
- Exon-by-exon sequencing of DNA or sequencing RT-PCR products is typically used to scan for mutations in a gene.
- Many methods exist for testing for specific sequence changes.
What to Test and Why
- DNA can be obtained from any nucleated cell sample.
- Clinical considerations (e.g., sample accessibility and quality) may influence sample selection.
- Other sources of DNA include blood cells, skin fibroblasts, bone marrow biopsies, prenatal samples (chorionic villus sampling, amniocentesis), and single cells from blastocysts.
- RNA or a functional test may be preferable in some cases (e.g., enzyme activity tests). Samples from the tissue showing gene expression are needed.
DNA or RNA?
- If scanning for unknown mutations, reverse transcriptase PCR (RT-PCR) offers advantages.
- RNA analysis has disadvantages, including handling sensitivity to degradation; the gene of interest might not be expressed, or might be present at a low level.
- Treating samples with translation inhibitors (e.g., puromycin) can sometimes help detect transcripts with premature termination codons missed through NMD.
Scanning a Gene for Mutations
- Most diseases display significant allelic heterogeneity, requiring scans across or near related genes to identify mutations.
- Sequencing (Sanger or next-generation sequencing) is the common choice of method, often exon-by-exon for genomic DNA, or sequencing RT-PCR products.
- The method used might depend on the size/amount of DNA available
A Gene Scanned by Sequencing
- Sanger sequencing (dideoxy or capillary electrophoresis) remains the gold-standard for mutation scans.
- Its alternative, next-generation sequencing, offers a higher throughput of sequencing, but analysis needs to consider sequencing volume.
Scanning Methods Based on Mismatches or Heteroduplexes
- Heteroduplexes can form when a PCR product is heated and then cooled slowly, resulting in a complex DNA strand mixture.
- Heteroduplex formation reveals sequence discrepancies that indicate a mutant DNA strand.
- These differences are exploited in some tests using non-denaturing polyacrylamide gel electrophoresis, dHPLC, and DGGE.
Scanning methods Based on Single-Strand Conformation Analysis
- The protein truncation test (PTT) is used to detect frameshifts, splice site or nonsense mutations creating premature stop codons.
- This is a method more specific to abnormal protein termination, and not always easy to perform
Microarrays
- Microarrays provide a single-operation method to scan for almost any gene mutation.
- Amplified cDNA or gene exons are hybridized to a microarray containing overlapping oligonucleotides corresponding to every part of the sequence.
- Arrays are typically optimized for specific diseases with limited allelic heterogeneity (e.g., BRCA1/2 or cystic fibrosis).
DNA Methylation Patterns
- DNA methylation patterns can be detected by various methods.
- Restriction enzyme digestion is often utilised to determine methylation patterns.
- Methylated DNA exhibits different patterns compared to non-methylated DNA when digested with specific enzymes.
- Alternatively, PCR is used for amplified DNA that undergoes digestion, allowing the detection of methylated or unmethylated sequences.
- Genome-wide methylation studies employ chromatin immunoprecipitation.
Unclassified Variants
- Characterizing variants can be difficult.
- Consider co-segregation with disease within a family to assess pathogenicity; variants arising de novo (parent-absent) in a candidate disease gene strongly suggest pathogenic mutations.
- Testing ethnically matched controls might also be helpful, though with limitations.
- Performing functional studies (e.g., evaluating RNA splicing effects if at all possible) and in-silico predictions of pathogenic effects help.
- Evaluating species conservation of the variant sequence is a relevant pointer for pathogenicity.
Testing for a Specified Sequence Change
- Directly testing for known alterations is more straightforward than a complete gene scan.
- Genotyping of samples can easily be performed via sequencing, but standard sequencing may not be suitable for single-nucleotide-position screening in all cases.
- PCR-based tests can evaluate well-known sequence changes.
- Restriction enzyme tests use a known restriction site and observe if a specific cut happens or not to identify mutations present in the sample.
- PCR mutagenesis can insert artificial restriction sites, allowing to test for presence of particular alterations not previously characterised.
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Description
Explore the key concepts of genetic testing in Chapter 18 of Human Molecular Genetics. This quiz covers the methodologies, importance of accuracy, and clinical considerations associated with genetic tests including DNA and RNA analysis. Test your understanding of how these techniques are applied in clinical settings.
