Basic Research In Preparation And Application Of Oligonucleotide Microarray For DPYD Genotyping

Although the application of 5-Fu is very wide to cancer patients and curative effect is notable, severe side-effects even death will happen sometimes. If we can predict its reaction before using 5-Fu, it will alleviate patients' suffering, change previous treatment pattern radically and realize individual medical treatment.Because more than 85% of 5-Fu is degradated by DPD, patients who have deficiency or descent in DPD activity will suffer severe side effects. At present, along with molecule biology's development and deeply understanding to DPYD, it is possible to predict reactions of 5-Fu in gene level. It is known the gene that encodes for DPD expression is called DPYD whose polymorphism on EXON2T85C(DPYD9) implies deficiency in DPD activity. So the patient with EXON2T85C(DPYD9) mutation could experience toxicity in response to 5-Fu therapy.The test predicted reaction of 5-Fu by detecting DPYD polymorphism on EXON2T85C(DPYD9) using gene chips directly. At the same time, we detected DPYD polymorphism on EXON13A1627G(DPYD5) for its higher mutation frequency in papers. Also the mutation frequency in Chinese population was detected on EXON2G62A(DPYD12),EXON13G1601A(DPYD4),EXON14deleteC1897(DPYD3) and EXON14intronl4GIA(DPYD2). Now DPYD gene chip has been prepared, and its some influence factors have been optimized, at one time, we have screened a lot of clinic samples by gene chips in Chinese (cancer patients, normal person, nephropathy patients) for the first time. 1 Preparation and optimization of DPYD gene chipWe searched important SNPS and its flanking sequences. Primers and probes were designed aidedly by computer then synthesized. DPYD gene chip was prepared byprinting the target DNA in bromine or aldehyde modified glass. Optimization of PCR and hybridization were excuted. Mjalty PCR was attempted. Repeated trial with plasmid showed hybridization results is credibility. In addition, effect of ultraviolet crosslinking energy was made on its hybridization.We constructed successfully standard template and constituted criteria for classification . 2 DPYD genechips' application in classifying of drug catabolic enzyme geneGenotyping results displayed the allele frequency on EXON2T85C(DPYD9) in Chinese (cancer patients,normal person,nephropathy patients) were 15/112,3/45,9/83, allele frequency on EXON13A1627G(DPYD5) were 40/112,14/45,23/83. At the same time, it were compared on EXON2T85C(DPYD9) and EXON13A1627G(DPYD5) in cancer patients and different gender, the allele frequency of EXON2T85C(DPYD9) in cancer patients was 6/72, it was 9/40 in other patients. Mutation frequency of EXON13A1627G(DPYD5) was 26/72 in cancer patients and was 14/40 in other patients. It was 11/70 in men and 4/42 in women for EXON2T85C(DPYD9). For EXON13A1627G(DPYD5 it were 25/70 and 15/42.On statistics we concluded the allele frequency on EXON2T85C(DPYD9) and EXON13A1627G(DPYD5) were not different obviously in Chinese.the difference was obvious on EXON2T85C(DPYD9) and not obvious on EXON13A1627G(DPYD5) in cancer and other patients.it was not evident on EXON13A1627G(DPYD5).these was not evident difference on EXON2T85C(DPYD9) and EXON13A1627G(DPYD5) in men and women.We also screened these alleles in these people by DPYD genotyping chips, such as EXON2G62A(DPYD12),EXON13G1601A(DPYD4),EXON14deleteC1897(DPYD3) and EXON14intronl4GIA(DPYD2),mutation is not found.17 PCR products were sequenced for proving veracity of genechip, 16 was accordant to its hybridization results, it is about 94.12%. it proved the results' reliability again.These results set the foundation to establish gene chips technique flatporm and develop individuation project in Chinese population. At the same time, it will play animportant role in proving the connection between DPYD polymorphism and clinicalreaction of 5-Fu.