Molecular genetics - Advancements in Genetic analysis (Ib) Flashcards
Limitation of traditional PCR?
- Non-automated, need processing
- Short dynamic range, not quantitative
- EB staining not sensitive
Principle of real time PCR
Advantages of real time PCR
Fluorescence-based detection, real-time monitoring of amplified product accumulation
Adv:
- Automatic detection and quantitative measurement
- Board dynamic range of detection
- High sensitivity
- High throughput and low cost
4 steps of RT-PCR
- Polymerization: Fluorescent reporter dye and quencher are attached to both ends of probe
- Stand displacement: when the probe is intact, the reporter dye emission is quenched
- Cleavage: During each extension cycle, the DNA polymerase cleaves the reporter dye from the probe
- Polymerization complete: Once separated from the quencher, the reporter dye emits its characteristic fluorescence
4 applications of RT-PCR
1) Quantitation of gene expression
2) DNA copy number variation analysis
3) Viral load quantitation
4) SNP genotyping
Digital PCR
- Principle
Combine limiting dilution + end-point PCR + Poisson statistics to get absolute measurement of nucleic acid concentration
- PCR sample is partitioned into droplet. Each droplet is a PCR reaction
- PCR droplets with target sequence = fluorescence positive
- PCR droplets without target sequence = no fluorescence
- Poisson statistical analysis of positive and negative droplets yield absolute quantitation of target sequence
Advantage of digital PCR
Adv:
- Detection of rare gene targets against wild type
- Detection of target at very low levels
- Detect small fold differences in targets
- No need to rely on references and standards
Applications of digital PCR
Low-level pathogen detection
Non-invasion prenatal test using cell-free DNA
Detect rare mutation in heterogeneous tumor sample
cDNA microarray
- Principle
- Thousands of genetic probes spotted onto glass slide
- cDNA labelled with fluorescence dye and hybridized to microarray
- Genome-wide analysis of gene expression/ gene expression profiler
e.g. Red dye = upregulation, green dye = down regulation, black = constitutive expression
Compare control and sample for differences in dyes
Most common type of genetic variant?
Effect?
Single nucleotide polymorphism (SNP)
Mostly occur in non-coding regions, no effect
SNP testing
- Principle
SNP used as tags to identify and locate disease associated genes/ chromosomal regions
SNP studied as marker of complex disease phenotype
SNP testing
- Applications (5)
- Paternity test
- Inheritance of disease gene in family
- Predict risk of disease development
- Predict drug response
- Predict susceptibility to environmental factors
2 SNP screening strategies?
1) Many SNP in few cases - discover novel disease associated SNP
2) Few SNP in many cases - common disease associated SNP, achieve high statistical power
Describe the TaqMan SNP genotyping assay
TaqMan SNP genotyping assay
Pair of PCR primers detect specific SNP targets
Pair of allele specific TaqMan probes contain distinctive fluorescent dyes
e.g.
Dye A only = homozygous for allele 1
Dye B only = homozygous for allele 2
Both Dye A and B signal = heterozygous for both allele
Test that detect SNP allele based on actual mass?
Massarray/ Mass spectrometry
Amplification > Primer extension > spin to separate by mass > detection and ratio analysis
Method for genome-wide SNP analysis?
SNP Microarray
DNA from blood or buccal swab
Label with fluorescence
Hybridize on microarray that contains genetic probes specific for 1 million SNPs
Purpose of GWAS
- Study thousands of SNPs at the same time by microarray
- Identify genetic variations that contribute to common and complex diseases
- Development of personalized medicine
Limitations of GWAS
- No info on disease mechanism
- Statistical correlation but no true disease prediction
- Need Large cohort size for meaningful statistical cohort
- Low discriminatory and predictive ability
Next generation sequencing
- Advantage
- High throughput sequencing: analyze >100million sequences in parallel
- Automatic, reduce manpower cost
- Minimize sequencing reagents, lower sequencing cost
Next generation sequencing
- Disadvantage
- Raw sequencing call accuracy (10x lower than Sanger)
- Limited read length (under 100bp only)
- Algorithmic and bioinformatics challenges
2 types of NGS at DNA level
Whole-genome (coding and non-coding DNA): e.g. somatic mutations and chromosome rearrangement in HBV, HCC
Whole-exome sequencing (coding exons DNA): somatic mutation or homozygous gene deletions
2 types of NGS at RNA level
Transcriptome sequencing (PolyA and mRNA) microRNA sequencing (Small size RNA)
2 types of NGS at epigenetics level
Whole genome bisulfite sequencing (DNA methylation)
Chromatin immuno-precipitation sequencing (Histone modification)
Applications for NGS?
- SNP discovery
- DNA copy number
- Chromosome translocation
- Mutation detection
- Viral DNA integration site mapping
- De novo Genome sequencing for new pathogens (e.g. COVID-19)
Application of single cell sequencing
- Stem cell research
- Circulating tumor cells
- Cancer heterogeneity
- Immune microenvironment
personalized medicine
- Principle
- Purpose?
Use biomarkers to classify individuals by susceptibility to disease or response to specific treatment
Risk prediction (e.g. adverse drug reaction, infection)
Disease diagnosis
Therapeutic decision
Manage prognosis
3 examples of personalized medicine in cancer treatment
1) HER2 overexpression in breast cancer: Tratuzumab
2) ALK rearrangement in lung cancer: Crizotinib
3) BRAF mutation in melanoma: Vemurafinib
