150 journal reprt

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

Single Nucleotide Polymorphism

• A type of single nucleotide alteration

• Useful GENETIC MARKERS for molecular diagnosis, prognosis, drug response, and predisposition to diseases

Droplet-allele specific-polymerase chain reaction Machine

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

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

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

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

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.

Results

• amplification was positive and determined the genotypes of the all 8 SNP loci within 8 min

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.

Results

• Droplet-PCR : 0.1–5.0% • 7900HT Genetic Analyzer: 0.5–5.0%; No

detection at rs430152 and rs2385512 loci

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

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

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.