Characterization of recombinant single-stranded DNA-binding protein from Escherichia coli and its application in accurate pyrosequencing / 生物工程学报
Chinese Journal of Biotechnology
; (12): 1513-1520, 2011.
Article
in Zh
| WPRIM
| ID: wpr-304550
Responsible library:
WPRO
ABSTRACT
We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (T(m)) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the T(m) of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the T(m) difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.
Full text:
1
Index:
WPRIM
Main subject:
Recombinant Proteins
/
Sequence Analysis, DNA
/
Diphosphates
/
DNA-Binding Proteins
/
Escherichia coli
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Genetic Vectors
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Genetics
/
Metabolism
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Methods
Language:
Zh
Journal:
Chinese Journal of Biotechnology
Year:
2011
Type:
Article