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

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. Single Nucleotide Polymorphism
• A type of single
nucleotide alteration
• Useful GENETIC
MARKERS for
molecular diagnosis,
prognosis, drug
response, and
predisposition to
diseases

3. Droplet-allele specific-polymerase
chain reaction Machine

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

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. Results
• Droplet-PCR : 0.1–5.0%
• 7900HT Genetic Analyzer: 0.5–5.0%; No
detection at rs430152 and rs2385512 loci

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. 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. 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.