生物学杂志

Journal of Biology ›› 2024, Vol. 41 ›› Issue (2): 103-.doi: 10.3969/j.issn.2095-1736.2024.02.103

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Establishment and application of a multiple allele-specific PCR method based on a novel high-fidelity Taq polymerase

This technology was established on the basis of three selected SNP loci for preliminary testing on mice with partially replaced fragments on chromosome 8, including specificity testing of high-fidelity Taq enzymes and evaluation of the effect of primer concentration on genotyping results as well as sensitivity assessment. It was used to detect nine SNPs in different wild-type chromosome 1 substitution mouse samples. The study showed that the detection results without introducing mutations into primers were consistent with sequencing results. The dilution experiments indicated that the optimal concentration was at least 0.003-0.006 μmol/L, while sensitivity detection revealed that the minimal concentration detection limit could reach below 0.07 ng/μL. Finally, five standard samples’ genotypes representing combinations of nine SNPs were detected. The overall process for applying this highly specific Taq enzyme in multiple allele-specific PCR experiments was eventually validated and established. This article offered a low-cost solution with high specificity and the ability to detect multiple SNP loci, which preserved the reference value for the development of multiple allele-specific PCR technology.   

  1. School of Biomedical Engineering, Donghua University, Shanghai 201620, China
  • Online:2024-04-18 Published:2024-04-17

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Abstract: This technology was established on the basis of three selected SNP loci for preliminary testing on mice with partially replaced fragments on chromosome 8, including specificity testing of high-fidelity Taq enzymes and evaluation of the effect of primer concentration on genotyping results as well as sensitivity assessment. It was used to detect nine SNPs in different wild-type chromosome 1 substitution mouse samples. The study showed that the detection results without introducing mutations into primers were consistent with sequencing results. The dilution experiments indicated that the optimal concentration was at least 0.003-0.006 μmol/L, while sensitivity detection revealed that the minimal concentration detection limit could reach below 0.07 ng/μL. Finally, five standard samples’ genotypes representing combinations of nine SNPs were detected. The overall process for applying this highly specific Taq enzyme in multiple allele-specific PCR experiments was eventually validated and established. This article offered a low-cost solution with high specificity and the ability to detect multiple SNP loci, which preserved the reference value for the development of multiple allele-specific PCR technology.

Key words: allele-specific PCR, multiplex PCR, high-fidelity Taq enzyme, genotype, chromosome substitution mice

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