Nanopore DNA single molecule sensing method and synthetic nanopore fabrication / 国际生物医学工程杂志

Nanopore single molecule sequencing(SMS) is considered as one of the most promising third generation DNA sequencing method by virtue of fast sequencing and low cost. As the most significant part of the sequencing system, the synthetic solid nanopore has recently been the research highlight in the field of nanopore DNA sequencing because of its fine-controlled size, high reliability and wide applicability. Also, its fabrication method is currently one of the main challenges in this field. In this article, the principles of the nanopore SMS are introduced followed by the review of the difficulties and challenges in the current research stage with emphasis on the nanopore fabrication methods.

Rapid purification, characterization and substrate specificity of heparinase from a novel species of Sphingobacterium.

A type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock. It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold. MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75,674 Da of the native enzyme. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. K(m) and V(max) of the heparinase for de-o-sulfated-N-acetyl heparin were 42 micro M and 166 microM/min/mg protein, respectively. The heparinase showed similar activity on both heparin and heparan sulfate, except for the heparin from bovine lung. The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4%. Thus modification of N-site in heparin structure was favorable for heparinase activity. On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150%. Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources.