Nucleic Acids (DNA) Sequencing, Transcriptional

Abstract

Transcriptional Sequencing (TS) is a novel DNA sequencing method based on the chain termination method using RNA Polymerase (RNAP). Transcriptional sequencing differs considerably from conventional cycle sequencing methods using DNA Polymerases (DNAPs) with 2′-deoxyribonucleotide triphosphates (2′-dNTPs) and 2′, 3′-dideoxyribonucleotide triphosphates (2′,3′-ddNTPs). The TS method employs a modified T7, T3, or SP6 RNAP with ribonucleotide triphosphates (rNTPs) and 3′-deoxyribonucleotide triphosphates (3′-dNTPs) as substrates and terminators, respectively, and the T7, T3, or SP6 promoter sequence is used as initiation site instead of the primer sequence. The characteristics of RNAP allow TS to be conducted under conditions that are substantially different from conventional cycle sequencing using DNAP. In TS, the reaction is performed isothermally (at 37°C) and is stable with high processivity. Transcriptional sequencing can be applied to various amounts of double-stranded DNA template because RNAP produces a large number of transcribed RNA molecules and the quantity of product is limited by the quantity of substrate rather than that of the template. Furthermore, in direct sequencing using TS, PCR products do not need to be purified before the sequencing reaction because RNAP requires neither 2′-dNTPs nor single-stranded PCR primers, but polymerization is primed using specific double-stranded promoters and rNTPs. This characteristic feature ensures that even highly GC-rich DNA templates can be sequenced effectively. Transcriptional sequencing is also applicable to MALDI-TOF-MS DNA sequencing owing to the stability of the RNA polymer against fragmentation caused by laser ablation. Thus, TS differs considerably from conventional cycle sequencing methods using thermostable DNA polymerases, but should complement such methods in numerous practical applications. Keywords: RNAP Mutant (in TS); MALDI-TOF-MS Sequencing; TS