Practical application of transcriptional sequencing for GC-rich templates.
Transcriptional sequencing (TS) is a method that differs considerably from conventional sequencing methods. These differences include the use RNA polymerases with rNTPs and 3'-dNTPs as substrates and terminators respectively, and initiation from double stranded promoters on templates of ds-DNA. We used TS in an attempt to sequence 33 clones whose electropherogram peaks suddenly became absent or weak with conventional sequencing methods. All of the TS reactions overcame the difficulty in sequencing the problematic target regions of the 33 clones. Therefore, TS can be applied to sequence not only GC-rich regions, but also whole genome sequences with a high GC content.
Shibata
Kazuhiro
K
Genome Science Laboratory, RIKEN Discovery and Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. rgscerg@gsc.riken.go.jp
Izawa
Masaki
M
Hayashizaki
Yoshihide
Y
Watahiki
Masanori
M
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004586:Electrophoresis; D020862:GC Rich Sequence; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D004247:DNA; D012321:DNA-Directed RNA Polymerases