Development of high performance chromatography-based gene detection system

2003 Fiscal Year Final Research Report Summary

• Project/Area Number: 14550739
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 化学工学一般
• Research Institution: Yamaguchi University
• Principal Investigator: YAMAMOTO Shuichi Yamaguchi University, Faculty of Engineering, Professor, 工学部, 教授 (80144921)
• Co-Investigator(Kenkyū-buntansha): MIYAGAWA Eiji Fuji Rebio, Senior Researcher, 検査薬開発部, 主任研究員 ; HINODA Yuji Yamaguchi University, School of Medicine, professor, 医学部, 教授 (10165128)
• Project Period (FY): 2002 – 2003
• Keywords: DNA / chromatography / biorecognition / melt curve / conformational changes / SNPs / electrostatic interaction / size exclusion curve

Research Abstract

There is an increasing need for rapid and quantitative analysis for DNA, especially for SNP (single nucleotide polymorphism) detection. Although DNA chips or microarrays are considered to be very promising detection methods, chromatography is also suited for this purpose as fully automated HPLC systems are already available. However, biorecognition in chromatography of SNPs is not well understood compared with that of other biomolecules. In this study the biorecognition mechanism in electrostatic interaction chromatography (ion-exchange chromatography, IEC) of DNA was investigated on the basis of our model proposed for IEC of proteins. Linear gradient elution experiments were carried out for various oligonucleotides as samples. The peak salt concentration I_R was measured as a function of normalized gradient slop GH. From the GH I_R curves, the number of binding sites B was determined. There was a good correlation between B and the length of oligonucleotides (or the number of negative charges). Th B values did not change with the mobile phase pH. This is different from IEC of proteins where B values strongly depend on the mobile phase pH. The I_R value increased with increasing molecular weight of oligonucleotides. Although small oligonucleotides having a single nucleotide difference can be easily resolved by conventional gradient elution IEC, another new efficient method is needed for larger oligonucleotide separation (SNPs detection)