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Escherichia coli single‐stranded DNA‐binding protein, a molecular tool for improved sequence quality in pyrosequencing
Author(s) -
Ehn Maria,
Ahmadian Afshin,
Nilsson Peter,
Lundeberg Joakim,
Hober Sophia
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200210)23:19<3289::aid-elps3289>3.0.co;2-#
Subject(s) - pyrosequencing , biology , primer (cosmetics) , dna , complementary dna , computational biology , library , dna sequencing , template , microbiology and biotechnology , genetics , gene , chemistry , nanotechnology , 16s ribosomal rna , materials science , organic chemistry
Pyrosequencing is a four‐enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single‐nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single‐stranded DNA‐binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real‐time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer‐annealing efficiency have also been evaluated.