Open AccessAb initio DNA synthesis by Bst polymerase in the presence of nicking endonucleases Nt.AlwI, Nb.Bbv CI , and Nb.BsmI
Author(s) -
Antipova Valeriya N.,
Zheleznaya Lyudmila A.,
Zyridezhda V.
Publication year - 2014
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/1574-6968.12511
Subject(s) - palindromic sequence , dna , palindrome , endonuclease , dna synthesis , recognition sequence , restriction enzyme , dna polymerase , nucleotide , biology , ab initio , polymerase , chemistry , genetics , gene , genome , organic chemistry
In the absence of added DNA, thermophilic DNA polymerases synthesize double‐stranded DNA from free dNTP s, which consist of numerous repetitive units ( ab initio DNA synthesis). The addition of thermophilic restriction endonuclease (REase), or nicking endonuclease (NEase), effectively stimulates ab initio DNA synthesis and determines the nucleotide sequence of reaction products. We have found that NEases Nt.AlwI, Nb.BbvCI, and Nb.BsmI with non‐palindromic recognition sites stimulate the synthesis of sequences organized mainly as palindromes. Moreover, the nucleotide sequence of the palindromes appeared to be dependent on NEase recognition/cleavage modes. Thus, the heterodimeric Nb.BbvCI stimulated the synthesis of palindromes composed of two recognition sites of this NEase, which were separated by AT‐reach sequences or (A) n (T) m spacers. Palindromic DNA sequences obtained in the ab initio DNA synthesis with the monomeric NEases Nb.BsmI and Nt.AlwI contained, along with the sites of these NEases, randomly synthesized sequences consisted of blocks of short repeats. These findings could help investigation of the potential abilities of highly productive ab initio DNA synthesis for the creation of DNA molecules with desirable sequence.