The effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I

Abstract

In the presence of the Nt.BspD6I nicking endonuclease DNA polymerase Bst stimulates intensive template/primer-independent DNA synthesis. Template/primer-independent DNA synthesis could be the reason for appearing nonspecific DNA products in many DNA amplification reactions particularly in the reactions with using nicking endonucleases. Search of the modes for inhibition template/primer-independent DNA synthesis becomes an urgent task because of broadening the DNA amplification methods with using nicking endonucleases. We report here that the E. coli single-stranded DNA binding protein has no effect on the template/primer-independent DNA synthesis. In the absence of the nicking endonuclease the single-stranded DNA binding protein encoded by bacteriophage T4 gene 32 completely inhibits template/primer-independent DNA synthesis. This protein does not inhibit synthesis of specific DNA product in the presence of nicking endonuclease but remarkably decreases the amount of nonspecific products.

Enhancing-effect of gold nanoparticles on DNA strand displacement amplifications and their application to an isothermal telomerase assay

“…This kind of non-specic reaction has not been well understood and lacks efficient suppressing methods, resulting in a major impediment to the practical applications of the NE-SDA methods. 13 A recent report found that the single-stranded DNA binding protein T4 bacteriophage gene 32 (T4gp32) could inhibit the RE-pol DNA synthesis; 37 our observation proved that AuNPs might also be inhibitors to this poorly-understood non-specic reaction. Also, thanks to the sensitive response of the surface plasmon of AuNPs to the environment, the interaction between AuNPs and the reaction components was monitored by UV-Vis spectroscopy, which aided in further understanding the way AuNPs inuence the amplication reactions and in tuning the concentration of AuNPs.…”

Ab initioDNA synthesis by Bst polymerase in the presence of nicking endonucleases Nt.AlwI, Nb.BbvCI, and Nb.BsmI

“…This preference may be manifested by palindromic sequences, as well as a number of other peculiarities. Thus, an increased temperature significantly increased GC content of synthesized DNAs (Ogata & Miura, ); the addition of SSB proteins affected the efficiency of the synthesis (Zyrina et al ., ), whereas the presence of REases resulted in the appearance of their recognition sites in the reaction products (Liang et al ., ). However, relative distribution of these sites and their orientation as well as the context and the size of spacer regions are poorly predictable.…”

“…This phenomenon is significant in studies of the possibilities for the direct creation of genetic information by a protein and for an understanding of possible mechanisms of genome evolution. The ab initio DNA synthesis is considered one of the causes of the appearance of nonspecific products in isothermal DNA amplification reactions (Inoue et al ., ; Tan et al ., ; Zyrina et al ., ; He & Jiang, ).…”

Ab initiosynthesis by DNA polymerases

“…Such a new strategy was offered for isothermal DNA amplification in the presence of nicking enzymes (Zyrina et al, 2012). It was based on the use of SSB proteins as inhibitors of non-specific ab initio synthesis.…”