Improvements in Pyrosequencing technology by employing Sequenase polymerase.
Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.
Gharizadeh
Baback
B
Stanford Genome Technology Center, Stanford University, 855 California Avenue, Palo Alto, CA 94304, USA.
Eriksson
Jonas
J
Nourizad
Nader
N
Nordström
Tommy
T
Nyrén
Pål
P
D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase; D011756:Diphosphates; D009690:Nucleic Acid Conformation; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D011756:Diphosphates; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase