[Simultaneous detection of polymorphisms in the 3'- and 5'-UTR of thymidylate synthase gene using denaturing high-performance liquid chromatography].
Polymorphism which is either in the thymidylate synthase (TS)enhancer region (TSER) of the 5-primer untranslation region (5' UTR) or in the 3-primer untranslation region (3' UTR) has been reported to be associated with the alterations in TS mRNA and protein levels. The TSER is characteristic of the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphism (SNPs) in 3R, as well as the polymorphism of the fragment length(FLP) in the TS 3'-UTR which is characteristic of the presence or absence of a 6 bp- nucleotide fragment, has recently been reported to be associated with the response to 5-fuorouracil (5-FU)-based chemo-therapy. The aim of the present study was to develop a specifc denaturing high-performance liquid chromatography (DHPLC) method for the rapid and simultaneous detection of these variations in clinical samples. Multi-PCR primers were designed to amplify the two regions simultaneously, The 8.6 min DHPLC gradient was optimized to include the analysis of multiplexed TSER/3' UTR chromatogram peaks, allowing for the simultaneous detection of 28 bp VNTRs and 6 bp FLP under nondenaturing conditions (50). The optimal melting temperature was determined experimentally for the detection of SNP in the TSER VNTRs. Finally, the DHPLC analysis was verified in parallel with PCR-RFLP and sequencing. The optimized DHPLC method resolved 100% of the known TS variations, discriminated between homozygous and heterozy-gous genotypes. This developed DHPLC method could permit the rapid, sensitive, and accurate identification of the TS genotypes (VNTRs, SNPs, and FLP).
Zhang
Xiao-Mei
XM
Jiangsu Institute of Cancer Research, Nanjing, China. zxm901@yahoo.com.cn
Chen
Sen-Qing
SQ
Cao
Hai-Xia
HX
Wu
Jian-Zhong
JZ
Ma
Guo-Jian
GJ
Li
Jin-Tian
JT
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D005838:Genotype; D006801:Humans; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism; D020506:Untranslated Regions / Q000235:genetics*
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D020506:Untranslated Regions; D013940:Thymidylate Synthase