Method of DNA synthesis
The present invention relates to an in vitro cell-free process for production of deoxyribonucleotides (DNAs) comprising at least one hairpin, corresponding DNA products and uses thereof, and oligonucleotides and kits useful in the process of the invention.
1 . An in vitro cell-free process for production of a deoxyribonucleic acid (DNA) molecule which comprises a desired DNA sequence, the process comprising:
(a) contacting an immobilised oligonucleotide on a solid support with a series of template oligonucleotides which overlap in sequence, in the presence of at least one DNA polymerase under conditions promoting template-dependent extension of the immobilised oligonucleotide to produce a first DNA strand which comprises the desired DNA sequence and further comprises a first portion of a protelomerase target sequence proximal to the solid support, wherein the first DNA strand is a single-stranded molecule;
(b) introducing a DNA sequence comprising a second portion of the protelomerase target sequence into a distal end of the first DNA strand, such that the first and second portions of the protelomerase target sequence thereby create a complete protelomerase target sequence proximal to the solid support; and
(c) contacting the complete protelomerase target sequence proximal to the solid support with a protelomerase, to thereby produce and release the DNA molecule from immobilisation.
2 . The process according to claim 1 , wherein the first portion of the protelomerase target sequence is incorporated into the immobilised oligonucleotide prior to step (a).
3 . The process according to claim 1 , wherein step (a) includes incorporating the first portion of the protelomerase target sequence into the first DNA strand during the template-dependent extension of the immobilised oligonucleotide.