150 journal reprt


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Novel high-speed droplet-allele specific-polymerase chain reaction: Application in the rapid genotyping of single nucleotide polymorphisms

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150 journal reprt

  1. 1. 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
  2. 2. Single Nucleotide Polymorphism • A type of single nucleotide alteration • Useful GENETIC MARKERS for molecular diagnosis, prognosis, drug response, and predisposition to diseases
  3. 3. Droplet-allele specific-polymerase chain reaction Machine
  4. 4. 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
  5. 5. Fluorescence Detection: Real-time PCR PROBE MOLECULE F: Fluorescent Marker Q: 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
  6. 6. 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
  7. 7. 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
  8. 8. 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.
  9. 9. Results • amplification was positive and determined the genotypes of the all 8 SNP loci within 8 min
  10. 10. 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.
  11. 11. Results • Droplet-PCR : 0.1–5.0% • 7900HT Genetic Analyzer: 0.5–5.0%; No detection at rs430152 and rs2385512 loci
  12. 12. 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
  13. 13. 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
  14. 14. 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.
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