Optimization of transcription for mass spectrometric DNA sequencing = 질량분석에 의한 염기서열 결정용 전사반응의 최적화

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It is demonstrated here that a DNA segment can be sequenced by transcription reactions of the Y639F mutant T7 RNA polymerase. Addition of 2``-deoxyribonucleoside 5``-triphosphate (dNTP) and 2``, 3``-dideoxyribonucleoside 5``-triphosphate (ddNTP) instead of ribonucleoside 5``-triphosphate (NTP) and 3``-deoxyribonucleoside 5``-triphosphate (3``-dNTP) in the standard transcription reactions produce sets of base-specifically terminated transcripts. The conditions for the transcriptional sequencing reactions with Y639F mutant RNA polymerase were optimized for higher yields of transcripts. The optimum concentrations of terminating nucleotides (ddNTP) and elongating nucleotides (GTP, ATP, UTP, dCTP) were different for different sizes of RNA. The higher concentration of terminating nucleotide or the lower concentration of elongating nucleotide was, the more the shorter transcripts but the less the longer transcripts were produced. The order of the optimum ratio of "terminating nucleotide concentration" to "elongating nucleotide concentration" for higher yield of RNA transcripts was ddATP/ATP>ddGTP/GTP>ddTTP/UTP>ddCTP/dCTP, which was comparable to the relative selectivity of Y639F mutant for 3``-dNTP and NTP (3``-dATP/ATP<3``-dGTP/GTP, 3``-dUTP/UTP<3``-dCTP/CTP). This strategy would help difficulties arise due to the small mass difference (1Da) of U and C, and commercially not available 3``-dNTP, so that it can contribute to the mass spectrometric analysis of a single-tube transcription reaction for DNA sequencing.
Advisors
Kang, Chang-Wonresearcher강창원researcher
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
2001
Identifier
165811/325007 / 000993551
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물과학과, 2001.2, [ iv, 66 p. ]

Keywords

DNA sequencing; 염기서열결정

URI
http://hdl.handle.net/10203/27935
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=165811&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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