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Novel high-speed droplet-allele specific-polymerase chain reaction: Application in the rapid genotyping of single nucleotide polymorphisms
Presented by: Guevarra; Olivar; Santos, J.; Tan; Virata
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Single Nucleotide Polymorphism
• A type of single nucleotide alteration
• Useful GENETIC MARKERS for molecular diagnosis, prognosis, drug response, and predisposition to diseases
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Droplet-allele specific-polymerase chain reaction Machine
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Reaction Tube
For RAPID TEMPERATURE TRANSITION; allows the droplet of PCR mixture to move easily in the tube during the mechanical rotation of the machine with the 2 connected heater blocks.
Rotation of the PCR Machine
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Fluorescence Detection: Real-time PCRPROBE MOLECULEF: Fluorescent MarkerQ: Quencher Molecule; inhibits fluorescence by absorbing emission of F whenever F & Q are together in a strand
Binding of primer and probe and the subsequent action of the DNA polymerase
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Fluorescence Detection: Real-time PCR
Exonuclease activity of the DNA polymerase separating F & Q
Eventual detection of fluorescence and correlation of intensity of fluorescence with the number of amplicons present
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Methodology: Measuring the Efficiency of the Device
• Reactivity of the droplet-PCR using SNP genotype-specific primers and probes
• Detection limit of droplet-AS-PCR for 8 SNP loci
• Rapid SNP genotyping by droplet-AS-PCR using buccal cells
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Reactivity of the droplet-PCR using SNP genotype-specific primers and probes
• Source: Peripheral blood (PB) from 7 healthy volunteers with 8 SNP LOCI
• PCR cocktail (10 μL): – Allele-specific primers: SNP-matched
nucleotide in the 3 -end and a template ′mismatched nucleotide at the −2 position from the 3 -end (800nmol/L) ′ *
– TaqMan probe: fluorescein amidite at the 5 -′end nucleotide and quencher (Black Hole Quenchers) at the 3 -end nucleotide ′(300nmol/L)
– Platinum Taq DNA polymerase – Reaction buffer: Tris–HCl pH 9.0, KCl and MgCl2
• PCR Cycle: 95 °C for 10 s and 35 cycles at 95 °C for 4 s and 58–66 °C for 8 s.
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Results
• amplification was positive and determined the genotypes of the all 8 SNP loci within 8 min
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Detection limit of droplet-AS-PCR for 8 SNP loci
• Mixture of genomic DNAs having the homozygote genotype (AA, GG, CC, or TT) with 5-fold serial dilution of genomic DNAs having the alternate homozygote genotype (GG, AA, TT, or CC), or vice versa
• Serial dilutions were prepared using a 50 ng/μL starting concentration.
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Results
• Droplet-PCR : 0.1–5.0% • 7900HT Genetic Analyzer: 0.5–5.0%; No
detection at rs430152 and rs2385512 loci
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Rapid SNP genotyping by droplet-AS-PCR using buccal cells
• Source: Buccal cells from 5 healthy volunteers• PCR cocktail: 20 mg/mL proteinase K in lysis
buffer (50 mmol/L Tris–HCl pH 8.5, 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Tween-20, 0.5% NP40, 20 mmol/L DTT) – 50 °C for 1 min, and then denatured at 95 °C for 1
min to inactivate the proteinase • No DNA extraction
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Result
• All the 8 SNP loci, and the genotypes at the 8 SNP loci were determined within 9 min when the fluorescence level of the amplification was over 6.7
• All the genotypes at 8 SNP loci determined by the droplet-AS-PCR assay were in accordance with that determined by direct sequencing
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Conclusion
• Droplet-AS-PCR can determine genotypes within 9 min while retaining specificity and sensitivity
• Buccal cells (not subjected to DNA extraction) were suitable samples for droplet-AS-PCR
• Droplet-AS-PCR provided rapid detection of single nucleotide alterations, including SNPs and mutations.