Allele-specific PCR with a novel data processing method based on difference value for single nucleotide polymorphism...

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Allele-specific polymerase chain reaction (AS-PCR) is an economical genotyping method but requires extensive optimization and time for result assessment.
• Existing AS-PCR methods face challenges in efficient parameter optimization and rapid result interpretation.

Purpose of the Study:

• To develop a novel data processing strategy for AS-PCR to overcome limitations in optimization and result assessment.
• To enable rapid and accurate single nucleotide polymorphism (SNP) genotyping.

Main Methods:

• AS-PCR combined with endpoint electrochemical detection using screen-printed electrodes.
• Simultaneous reactions with wild-type and mutant allele-specific primers.
• Calculation of the difference in redox current signals between two systems for genotyping.
• Real-time fluorescent PCR utilized for optimizing reaction parameters.

Main Results:

• Genotyping results obtained within 50 minutes.
• Analysis of 36 hair-root samples for ALDH2 gene genotypes showed complete agreement with commercial sequencing.
• The novel method successfully differentiated alleles using the difference value between two reaction systems.

Conclusions:

• The developed method provides a novel data processing approach for AS-PCR, significantly improving analysis speed.
• This technique is applicable for quick SNP analysis in health monitoring, disease prevention, and personalized medicine.