Multiple group-specific sequencing primers for reliable and rapid DNA sequencing.
Pyrosequencing technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.
Gharizadeh
Baback
B
Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology (KTH), Roslagstullbacken 21, SE-106 91, Stockholm, Sweden.
Ohlin
Andreas
A
Mölling
Paula
P
Bäckman
Anders
A
Amini
Bahram
B
Olcén
Per
P
Nyrén
Pål
P
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D020459:Genes, rRNA; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S