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Development of duplex-crossed allele-specific PCR targeting of TPMT*3B and *3C using crossed allele-specific blockers to eliminate non-specific amplification

Qu, Xue-Mei, Zhao, Na, Mo, Qin-Yun, Yao, Pu, Su, Ning, Wei, Kun, Wang, Liu, Huang, Jun-Fu, Ren, Xiao-Dong, Ren, Sai, Fu, Wei-Ling, Huang, Qing
Analytical biochemistry 2019 v.575 pp. 54-62
DNA-directed DNA polymerase, adverse effects, fluorescence, heterozygosity, loss-of-function mutation, nationalities and ethnic groups, polymerase chain reaction, therapeutics, thermodynamics, thiopurine S-methyltransferase, toxicity
Prospective testing for variants in the thiopurine S-methyltransferase (TPMT) is considered a key process in the development of thiopurine therapy. This testing is done to avoid toxicity and side effects in the management of diverse immunological and malignant conditions. Real-time fluorescent PCR techniques using duplex-crossed allele-specific primers in a single tube (DCAS-PCR) were developed in this study to genotype the common loss-of-function TPMT*3B c.460G > A (rs1800460) and TPMT*3C c.719A > G (rs1142345) usually occurring in individuals of Chinese ethnicity. In this method, several integrated strategies were used to completely eliminate the non-specific amplification that is commonly presented in traditional allele-specific (AS) PCR. These strategies include using AS-primers (ASP) that both are artificially mismatched in the penultimate positions and phosphorothioate modifications in the 5′-termini positions. In the assay, an AS-blocker was used, locus-specific TaqMan (LST) probes were used and we used at least two fragments were simultaneously amplified in a single tube which satisfy the thermodynamic characteristics of DNA polymerase to eliminate non-specific amplification. In a group of 200 unselected subjects, the results showed that 8 samples were heterozygous of TPMT*3C, and all samples possessed wild-type TPMT*3B. There was no non-specific amplification, and the genotypes were 100% consistent with Sanger sequencing.