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.