Methods of genotyping Flashcards
What does PCR-RFLP stand for?
Polymerase Chain Reaction - Restriction Fragment Length Polymorphism
It is a method that uses PCR followed by a restriction digest to differentiate between alleles.
What is the purpose of allele-specific PCR?
To amplify specific alleles of interest
This method ensures that only the desired allele is amplified for further analysis.
What is the significance of the T341C mutation in genotyping?
It removes the DdeI restriction site, resulting in a larger PCR product
This mutation can be used to test a polymorphism that affects the restriction enzyme.
Describe the TaqMan system.
A widely used automated allele-specific PCR system that employs fluorescent probes
The system detects fluorescence in real-time during PCR reactions.
What is MassARRAY technology used for?
Genotyping and mutation analysis
It utilizes MALDI-TOF mass spectrometry to identify genetic variations.
What are the advantages of SNP genotyping by Pyrosequencing?
Real-time detection of base incorporation and light measurement
It provides strong signals for homozygous and reduced signals for heterozygous genotypes.
What does GWAS stand for?
Genome Wide Association Studies
GWAS identifies associations between genetic variants and traits or diseases.
What is the typical significance threshold in GWAS?
5 x 10^-8
This threshold corrects for multiple testing in association studies.
What is the main technique used in manual DNA sequencing?
Sanger sequencing
This method uses chain termination and gel electrophoresis to determine DNA sequences.
What is a disadvantage of manual DNA sequencing?
Time-consuming and costly for large projects
Its limited throughput makes it inefficient for whole-genome sequencing.
What is exome sequencing?
A technique that focuses on sequencing only the exons of the genome
Exome sequencing targets 1-2% of the genome containing most disease-causing mutations.
What are the steps involved in whole genome sequencing?
Sample preparation, library preparation, sequencing, and data analysis
Each step is crucial for obtaining accurate genomic information.
What is the main advantage of whole genome sequencing?
Comprehensive coverage of all genetic variants
It captures variants in both coding and non-coding regions of the genome.
Fill in the blank: The Y axis in a typical GWAS Manhattan Plot represents the negative log of the _______.
p value
This indicates the statistical significance of each genetic variant.
True or False: High throughput sequencing can sequence fragmented DNA directly.
True
This technology allows for rapid sequencing of large sample sets.
What are the two main types of sequencing methods used in whole genome sequencing?
- Short-read sequencing
- Long-read sequencing
Each type has its own advantages and limitations regarding accuracy and read length.
What is the purpose of hybridization-based methods in exome sequencing?
To isolate exonic regions from the DNA library
These methods enrich the library for regions of interest.
What is the role of adapters in library preparation for sequencing?
To facilitate amplification and sequencing of DNA fragments
Adapters also allow for sample indexing in multiplexing.
What does the term ‘linkage disequilibrium’ refer to in GWAS?
The non-random association of alleles at different loci
It allows for the detection of associations with genes some distance away from the marker.
What is the main disadvantage of high throughput sequencing?
Data analysis challenges due to large volumes of data
Requires robust computational resources and expertise to interpret the results.
What is the significance of the negative log of the p value on the Y axis of a Manhattan Plot?
Indicates stronger statistical evidence of association with lower p values
Larger values signify more significant genetic variants.
List the steps of the Sanger sequencing process.
- DNA template preparation
- Reaction setup
- DNA synthesis and termination
- Fragment separation
- Detection
- Sequence analysis
Each step is essential for accurate DNA sequencing.
What is the primary method used for variant calling in whole genome sequencing?
Identifying SNPs, insertions, deletions, and structural variants
Variant calling is crucial for understanding genetic differences.
What are the main components of the sequencing by synthesis (SBS) method?
- Sample preparation
- Cluster generation
- Sequencing by synthesis
- Data analysis
SBS is a key technology in next-generation sequencing.
What does WGS stand for?
Whole Genome Sequencing
WGS is a comprehensive method for analyzing the entire genetic makeup of an organism.
What is a key advantage of Whole Genome Sequencing?
Comprehensive Coverage
It captures all genetic variants, including those in non-coding regions.
What is one flexible application of WGS?
Useful for both discovery and targeted research
WGS can be applied to explore new genetic insights as well as specific genetic inquiries.
Why is the data from WGS considered long-term relevant?
The data can be re-analysed as new insights and tools become available
This allows for ongoing exploration and understanding of genetic information.
What is a primary disadvantage of Whole Genome Sequencing?
Cost
WGS is more expensive than targeted approaches like exome sequencing.
What challenge is associated with the data volume generated by WGS?
Requires robust computational infrastructure
The massive datasets generated necessitate advanced computing resources for analysis.
What interpretation challenge arises from WGS?
Non-coding regions are less well-understood
This complexity makes it difficult to interpret the genetic data accurately.
Fill in the blank: WGS focuses on regions of interest, such as _______.
disease-associated genes
These genes are often linked to various phenotypic effects.
