A multiplexer liquidchip technology for detecting single nucleotide polymorphisms from metabolism of anti-thrombotic drugs in dried blood spots on filter paper.

Abstract

A single nucleotide polymorphism (SNP) is the most frequent type of variation in the genome. There are around 10 million SNPs that have been identified in the human genome [1]. Because SNPs are highly conserved throughout evolution and within a population, the map of SNPs serves as an excellent genotypic marker for research. The elucidation of SNP information will contribute to an individual’s susceptibility to disease and responsiveness to drug toxicity and medical intervention [2,3]. Nowadays, a variety of techniques have been used to perform SNP genotyping, but these techniques required whole blood as the sample. Dried blood spot (DBS) specimens require less material and are substantially more stable (several months at room temperature) than whole blood [4]. Thus, the simplicity of sample preparation, long time storage and convenient transport make DBS to be a cost-effective and suitable alternative tool for collecting blood sample. Plavix (clopidogrel) and warfarin are wildly used antithrombotic drugs in the treatment and prevention of thrombotic diseases, including myocardial infarction, ischemic stroke, and venous thrombosis [5]. However, because of genetic variation, the two drugs have wide inter-individual variation in dose resulting in the risk of serious bleeding complications [6,7].Many studies have shown that genetic status can greatly influence an individual patient’s warfarin or Plavix dosing [8–15]. So the Food and Drug Administration changed the warfarin label to encourage lower initial doses in patients who have the VKORC1–1639G.A, CYP2C9*2, or CYP2C9*3 allele in August 2007 [16] and added pharmacogenetic information to the Plavix product label because of genetic differences in CYP2C19 in March 2010. In this study, we developed a method to detect genotyping of DBS specimens about Plavix and warfarin using SurPlexTM-xTAGmethod. All oligonucleotides were synthesized by Invitrogen Corporation (Carlsbad, USA). PCR primers used for the genotyping assay (Table 1) were unmodified. Allele-specific primer extension (ASPE) primers consist of two sequences: a tag sequence (according to Luminex tag sequence) and the allele-specific sequence (Table 1). Universal anti-tags (probes) were amino (NH2)-modified for coupling to carboxylated microspheres. Tag sequences used for validation of the universal array were labeled with biotin. To evaluate the specificity of this method, the plasmids (pGEM-T-Easy; Promega, Madison, USA) containing wild-type (WT) alleles of CYP2C9, CYP2C19, VKORC1, and ABCB1 were constructed. The mutant-type plasmids were derived from WT plasmids using the TaKaRa Mutant Best kit (TaKaRa, Dalian, China). Approximately 30 ml of whole blood was spotted on 3-mm filter paper (Whatman, Wisconsin, USA). After drying, a 6-mm disk was cut out from the sampling paper. Additionally, the 6-mm disk was placed into a 1.5-ml Eppendorf cup (Hamburg, Germany), with 100 ml sterile water, and then placed in a boiling water bath for 15 min to make the cells lyse. After gentle vortexes, the mixture was centrifuged at 10,000 g for 5 min, then 10 ml supernatants were pipetted and used as DNA source for amplification. Multiplex PCR about warfarin was performed in a 50 ml volume containing: 1 Ex Taq polymerase buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.2 mM each of the primers, 1.0 U Ex Taq HS DNA polymerase (TaKaRa) and 10 ml supernatant of boiling DBS as template DNA. Thermo cycling was performed using 30 cycles of 958C for 30 s, 568C for 30 s, and 728C for 30 s. The reaction was concluded with a final extension step of 728C for 10 min and the product was kept at 48C until use. A similar approach was used to enrich DNA fragments of Plavix-related genes using 3 mM MgCl2. Exonuclease I and shrimp alkaline phosphatase (EXO-SAP) reaction was performed in a 25-ml volume containing: 1 SAP buffer, 1 U shrimp alkaline phosphatase (inactivating any remaining nucleotides) (TaKaRa), 10 U exonuclease I (degrading any remaining PCR primers) (New England Biolabs, Ipswich, USA) and 7.5 ml PCR product. Samples Acta Biochim Biophys Sin 2014, xx: 1–4 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu028.