2004 Fiscal Year Final Research Report Summary
Enzymatic Synthesis of Modified DNAs by PCR, and Their Application
| Project/Area Number |
13132202
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | Gunma University |
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Principal Investigator |
SAWAI Hiroaki Gunma University, Department of Applied Chemistry, Professor, 工学部, 教授 (70012648)
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| Co-Investigator(Kenkyū-buntansha) |
OZAKI Hiroaki Guama University, Department of Applied Chemistry, Professor, 工学部, 助教授 (90211820)
KUWAHARA Masayasu Gunma University, Department of Applied Chemistry, Research Associate, 工学部, 助手 (40334130)
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| Project Period (FY) |
2001 – 2004
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| Keywords | Nucleic Acid / Modified DNA / DNA polymerase / Modified Substrate / PCR / Enzymatic Reaction / Molecular Recognition / Aptamer |
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| Research Abstract |
If modified DNA can be prepared enzymatically by PCR from the substrate analogue using DNA polymerase, The resulting modified DNA could be used as a DNA probe by attachment of a reporter group, and as a DNA catalyst or as a DNA aptamer by in vitro selection. We prepared thymidine analogues bearing various functional groups at the C5 position, and examined whether they can be accepted as a substrate for the DNA polymerase. The functional groups studied in this research are an amino group, a carboxyl group, chelating groups, guanidine, imidazole, pyridine, various amino acids, fluorescein, acridone and sugars. We found that KOD Dash and Vent(exo-) DNA polymerases can incorporate many of these modified thymidine analogues and that the corresponding modified DNA can be obtained by PCR. On the other hand, no other DNA polymerases such as Taq DNA polymerase can accept these modified substrates. The resulting modified DNA shows no mutation at the modified portion. Similarly, cytidine analogues bearing several functional groups at C5 position can work as a substrate for PCR using the DNA polymerases. We also confirmed that modified DNA could be prepared by PCR from some modified adenine nucleotides. Further, combination use of modified thymidine, cytidine and adenine nucleotides for the PCR yielded the corresponding multi-modified DNA. We applied the modified DNA synthesis by PCR for the production of DNA aptamers using an in vitro selection technique. Modified DNA aptamers that bind specifically with sialllylactose that is present in a surface of cells, glutamic acid, a drug thalidomide or secondary and ternary structure of DNA have been developed. The sequence, secondary structure and binding affinity of the aptamers to the target compound have been studied to characterize the aptamers.
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