N.BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase

Abstract

Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.

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

“…We have previously shown that Bst DNA poly merase, similarly to many thermophilic polymerases, can synthesize DNA in the absence of template DNA and primer [5]. Therefore, the effect of SSB proteins on template/primer independent DNA synthesis per formed by Bst DNA polymerase in the absence of nicking endonuclease was studied first.…”

“…The choice of these timepoints was due to the fact that intense synthesis of high molecular weight DNA occurs mostly during the first hour of the reaction (at the DNA polymerase: nicking endonuclease ratio used in the present work). The synthesis is accompanied by DNA hydrolysis by nickase, since the DNA synthesized contains tandem sequences of nicking endonuclease recognition sites [5] (Fig. 3).…”

“…However, several consid erations on it are given below. According to the hypotheses concerning the mechanism of tem plate/primer independent DNA synthesis [3][4][5], for mation of hairpins priming further DNA synthesis is a decisive stage of the process. Stimulation of tem plate/primer-independent DNA synthesis by EcoSSB protein and suppression of the synthesis by the T4gp32 protein in the absence of nicking endonuclease can be accounted for by different geometry of EcoSSB/DNA and T4gp32/DNA complexes.…”

“…We have previously detected an acceleration of template/primer independent DNA synthesis in the presence of Nt.BspD6I nicking endonuclease [5]. The RUSSIAN [6] are increasingly used in many applications, including those developing for medical diagnostics.…”

Linear nicking endonuclease-mediated strand-displacement DNA amplification

“…In some cases, a brief period of specific amplification was followed by a rapid increase in nonspecific amplification. Thermophilic polymerases are known to be able to initiate template-independent de novo DNA synthesis and amplification [57,58], particularly in the presence of endonucleases [34,59,60]. We chose Sequenase 2.0 as the best polymerase candidate for further investigation because it has a far superior synthesis rate and processivity [55] compared to Klenow exo- and does not exhibit the nonspecific background amplification observed in most thermophilic polymerases.…”