Establishment Of The Targeted DNA Sequencing Analysis Method About The Anticancer Of Thiopurines

Thiopurine medication includes6-mercaptopurine(6-MP),6-thioguanine(6-TG)and its prodrug azathioprine(AZA).6-MP and6-TG are the two drugs currently usedin the threatment of acute lymphoblastic leukaemia(ALL) disease. AZA is used notonly in the ALL, but also mainly as immunosuppressive drugs used in theautoimmune diseases and after solid organ transplantation. Genetic polymorphisms insuch a key thioinosine monophosphate(TIMP) enzyme in thiopurine medicationmetabolism could lead to large interindividual variability in drug response and risk oftoxicity. Hence, the prospective identification of the frequency TPMT mutant alleles(TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C) could identify at higher risk ofdeveloping thiopurine-related adverse events.Establishment of the targeted DNA sequencing analysis method about theanticancer of thiopurines mainly include: Genomic DNA was extracted from patientsperipheral blood of the ALL disease and autoimmune disease. Primers Designed fromthe TPMT mutational sites, then multiple PCR amplification were conducted usingthem. Amplified products were subsequently analysed using agarose gel extraction.At last, purification DNA were sequenced from Applied Biosystems and TPMTmutant position were found through blasting them in NCBI.We applyed two ways to isolate the peripheral Blood Leukocytes: Red BloodCell lysis and Lymphocytes Separation medium. The latter could get the better result.The Blood Leukocytes were digested by proteinase K, extracted withPhenol/Chloroform and precipitated with ethanol.According to the TPMT mutant alleles (TPMT*2, TPMT*3A, TPMT*3B andTPMT*3C), we designed four pairs of primers. As a result, we found the Exon5,Exon7and Exon10to be useful. The optimized multiplex PCR reaction volumeswere as following: dNTP (10mM)4μL，Primers (20pmol)3μL (one primer1μL), TaqDNA polymerase2μL, and the total volume were50μl. All were supplemented with double distilled water. And the best annealing temperature and annealing time were55℃and60sec, respectively.As for the methods for PCR products purification, we gained that, the usualpurification method was the worst, the Agarose gels extraction took the second place,and the DNA Purification Kits was the best. The types of the TPMT could be got byblasting the PCR sequencing outcomes at the NCBI website.We used the above established analysis method to detect12ALL children. Onlyone TPMT*3C (A719G) could be found. When checked other9health people, no onemutational sites was found. This technique established a direct, high-effectgenotyping platform for analyzing and detecting, and it offered some new clues forclinical research.