Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes.
To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was approximately 10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts.
Dederich
Debra A
DA
Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor Plaza, Alkek Building, N1409, Houston, TX 77030, USA.
Okwuonu
Geoffrey
G
Garner
Toni
T
Denn
Amanda
A
Sutton
Angelica
A
Escotto
Michael
M
Martindale
Ashley
A
Delgado
Oliver
O
Muzny
Donna M
DM
Gibbs
Richard A
RA
Metzker
Michael L
ML
D000818:Animals; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016678:Genome; D005898:Glass; D008863:Microspheres; D010957:Plasmids / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
D004247:DNA