2005 Fiscal Year Final Research Report Summary
Development of small and massively-parallel fast DNA sequencer based on real-time monitoring of DNA replication reaction
| Project/Area Number |
16510155
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | Saitama University |
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Principal Investigator |
HUSIMI Yuzuru Saitama University, Graduate School of Science and Engineering, Professor, 理工学研究科, 教授 (80011641)
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| Project Period (FY) |
2004 – 2005
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| Keywords | DNA sequencing / ISFET / pH monitor / DNA replication / Surface reaction / Evolution reactor |
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| Research Abstract |
DNA sequencing for evolutionary molecular engineering and for SNP analysis requires a high-throughput sequencer for massive and closely related DNA samples. The sequencer that was developed here and based on real-time electrochemical monitoring of DNA replication reaction, is adequate for this purpose. Its basic principle is based on the adsorption of average 0.3 protons when single-nucleotide elongation reaction takes place at pH7. Following points were investigated. (1)Surface density of immobilized buoy DNA, which has thioU at the loop of loop-stem structure and bonded to the surface via S-S bond, were 1.3x10^4 molecules/μm^2. And the efficiency of hybridization of a sample DNA, which has a complementary sequence to buoy DNA and the region to be sequenced was about 70%, thus we get the sufficient density over the theoretical limit. (2)We confirmed the reusability of the buoy DNA through dissociation of the sample DNA. (3)We reconstructed the flow cell in which the differential two ISFETs were fixed, and observed the electric output from real-time pH increase due to 10 nucleotides elongation of the self-priming sample DNA. We observed the 8.5mV peak out put with the 10%-90% rise-time of 60s. Single nucleotide elongation will be able to be measured.
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