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
