| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 88 === In order to obtain 13C- and 15N-labeled deoxyribonucleotides, we followed and modified the procedure described by Smith et al. in 1997. Preparation of 13C- and 15N-labeled deoxyribonucleotides is necessary to resolve the highly overlapped proton spectrum. First, we obtained pGEX-2cdc8 plasmid from Dr. Su at Yang Ming University. This plasmid contains a cdc8 gene of the yeast Saccharomyces cerevisiae, which encodes the thymidylate kinase (TMPK). This enzyme can specifically phosphorylates dTMP to dTDP, and allow us to prepare in large scale of isotope-labeled dTTP. After transforming this plasmid and over expressing this protein in the host cell (E.coli BL21), we used glutahione-Sepharose column to purify this enzyme and then assayed its activity by HPLC method.
With 13C-methanol and 15N-ammonium sulfate as the sole carbon and nitrogen sources in the culture medium of M. methyltrophus, the bases and riboses of the DNA and RNA could be labeled with 13C/15N. The 13C- and 15N-labeled DNA and RNA were then isolated from the cell culture after being treated with RNase and isopropanol precipitation (instead of Affigel-601 column). More purified DNA could be obtained in this way. We then use DNase and Nuclease P1 to completely hydrolyze the DNA to 5'-monophosphate deoxyribonucleotides. Finally, 5'-monophosphate deoxyribonucleotides were enzymatically phsphorylated to 5'-triphosphate deoxyribonucleotides, which were then used to synthesize a defined DNA sequence by PCR.
|
|---|
