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Published on: September 25, 2018
Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single
Chiaki Taira1, Kazuyuki Matsuda, Akemi Yamaguchi
1Department of Laboratory Medicine, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
Background:
Single nucleotide alterations such as single nucleotide polymorphisms (SNP) and single nucleotide mutations are associated with responses to drugs and predisposition to several diseases, and they contribute to the pathogenesis of malignancies. We developed a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) with our droplet-PCR machine (droplet-AS-PCR).
Methods:
Using 8 SNP loci, we evaluated the specificity and sensitivity of droplet-AS-PCR. Buccal cells were pretreated with proteinase K and subjected directly to the droplet-AS-PCR without DNA extraction. The genotypes determined using the droplet-AS-PCR were then compared with those obtained by direct sequencing.
Results:
Specific PCR amplifications for the 8 SNP loci were detected, and the detection limit of the droplet-AS-PCR was found to be 0.1-5.0% by dilution experiments. Droplet-AS-PCR provided specific amplification when using buccal cells, and all the genotypes determined within 9 min were consistent with those obtained by direct sequencing.
Conclusions:
Our novel droplet-AS-PCR assay enabled high-speed amplification retaining specificity and sensitivity and provided ultra-rapid genotyping. Crude samples such as buccal cells were available for the droplet-AS-PCR assay, resulting in the reduction of the total analysis time. Droplet-AS-PCR may therefore be useful for genotyping or the detection of single nucleotide alterations.
Insights
A new droplet-based allele-specific PCR (AS-PCR) method allows for ultra-rapid genotyping of single nucleotide polymorphisms (SNPs) from crude samples like buccal cells. This assay demonstrates high specificity and sensitivity, reducing overall analysis time significantly.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Single nucleotide alterations, including single nucleotide polymorphisms (SNPs) and mutations, are crucial in drug response, disease predisposition, and cancer development.
- Allele-specific PCR (AS-PCR) is a method used for genotyping these alterations.
- Developing rapid and efficient genotyping assays is essential for clinical and research applications.
Purpose of the Study:
- To develop and evaluate a rapid genotyping assay using a droplet-based allele-specific PCR (droplet-AS-PCR) machine.
- To assess the specificity, sensitivity, and speed of the droplet-AS-PCR method for SNP genotyping.
- To determine the feasibility of using crude biological samples, such as buccal cells, without prior DNA extraction.
Main Methods:
- Developed a droplet-AS-PCR assay utilizing 8 SNP loci.
- Evaluated assay specificity and sensitivity through various experiments.
- Used buccal cells pretreated with proteinase K, bypassing traditional DNA extraction.
- Compared genotypes obtained from droplet-AS-PCR with those from direct sequencing.
Main Results:
- The droplet-AS-PCR assay demonstrated specific amplification for all 8 SNP loci tested.
- Sensitivity analysis revealed a detection limit ranging from 0.1% to 5.0% via dilution experiments.
- Genotyping of buccal cells using droplet-AS-PCR was consistent with direct sequencing results.
- The entire genotyping process was completed in an impressively short time of 9 minutes.
Conclusions:
- The novel droplet-AS-PCR assay provides high-speed amplification with retained specificity and sensitivity for ultra-rapid genotyping.
- The ability to use crude samples like buccal cells directly significantly reduces total analysis time.
- Droplet-AS-PCR is a promising tool for efficient genotyping and the detection of single nucleotide alterations in various settings.
Related Concept Videos
01:05Single Nucleotide Polymorphisms-SNPs
01:32PCR - Polymerase Chain Reaction