title: Pyrosequencing enhancement for better detection limit and sequencing homopolymers.
abstract: Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid and real-time sequence determination. Although ample genomic research has been undertaken using pyrosequencing, the requirement of relatively high amount of DNA template and the difficulty in sequencing the homopolymeric regions limit its key advantages in the applications directing towards clinical research. In this study, we demonstrate that pyrosequencing on homopolymeric regions with 10 identical nucleotides can be successfully performed with optimal amount of DNA (0.3125-5 pmol) immobilized on conventional non-porous Sepharose beads. We also validate that by using porous silica beads, the sequencing signal increased 3.5-folds as compared to that produced from same amount of DNA immobilized on solid Sepharose beads. Our results strongly indicate that with optimized quantity of DNA and suitable solid support, the performance of pyrosequencing on homopolymeric regions and its detection limit has been significantly improved.
authors.affiliation: Genome Institute of Singapore, 60 Biopolis Street, 02-01 Genome, Singapore 138672, Singapore.
authors.forename: Min
authors.initials: M
authors.lastname: Gong
authors.forename: Shi-Hui
authors.initials: SH
authors.lastname: Foo
authors.forename: Lifang
authors.initials: L
authors.lastname: Lin
authors.forename: Edison T
authors.initials: ET
authors.lastname: Liu
authors.forename: Baback
authors.initials: B
authors.lastname: Gharizadeh
authors.forename: Sanket
authors.initials: S
authors.lastname: Goel
mesh_terms: D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D011108:Polymers / Q000032:analysis / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D011108:Polymers; D004247:DNA