METHOD FOR PREPARING STABLE ISOTOPE-LABELED ssDNA BY BIOSYNTHESIS WITH ESCHERICHIA COLI

A method for preparing stable isotope-labeled single-stranded DNA (ssDNA) by biosynthesis with Escherichia coli (E. coli), comprising the steps of:

• 1) adding a site of a first restriction endonuclease and a site of a second restriction endonuclease to 5′ and 3′ ends of a target sequence, respectively, to obtain a repeating unit;
• wherein the first restriction endonuclease and the second restriction endonuclease are different;
• 2) ligating the repeating unit obtained in step 1) in tandem to obtain a fusion sequence;
• 3) inserting the fusion sequence in step 2) into a high-copy vector to obtain a recombinant vector;
• 4) introducing the recombinant vector in step 3) into host strain to obtain a recombinant E. coli strain;
• 5) extracting and purifying the recombinant vector from the recombinant E. coli strain after culture of the recombinant E. coli strain in step 4);
• where the culture is conducted in a medium with 15NH4Cl as a sole source of nitrogen and/or 13C-glucose as a sole source of carbon;
• 6) digesting the recombinant vector obtained in step 5) using the first restriction endonuclease and the second restriction endonuclease to obtain an asymmetric double-stranded DNA (dsDNA) structure, and isolating two ssDNAs of unequal lengths, with one of the two ssDNAs as a target sequence;
• and
• 7) recovering the target sequence in the two ssDNAs in step 6) to obtain the stable isotope-labeled target ssDNA.