08-02-2018 дата публикации
Номер: US20180037943A1
Принадлежит:
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 oligonucleotide immobilised 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 a first portion of a protelomerase target sequence proximal to the solid support;(b) introducing a DNA sequence comprising a second portion of the protelomerase target sequence into a distal end of the first DNA strand, or into a second DNA strand, such that the first and second portions of the protelomerase target sequence thereby create the protelomerase target sequence proximal to the solid support; and(c) contacting the 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 immobilised oligonucleotide of (a) comprises the first portion of the protelomerase target sequence.3. The process according to claim 1 , wherein the first portion of the protelomerase target sequence is introduced into the first DNA strand by template-dependent extension.4. The process according to claim 1 , wherein the DNA molecule is a single-stranded circular DNA molecule comprising at least one hairpin that comprises the first portion or the second portion of the protelomerase target sequence.5. The process according to claim 4 , wherein step (b) comprises template-dependent extension with one or more ...
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