US 11,913,050 B2
Scalable biotechnological production of DNA single strand molecules of defined sequence and length
Florian Praetorius, Munich (DE); and Hendrik Dietz, Haar (DE)
Assigned to TECHNISCHE UNIVERSITÄT MÜNCHEN, Munich (DE)
Appl. No. 16/329,495
Filed by TECHNISCHE UNIVERSITÄT MÜNCHEN, Munich (DE)
PCT Filed Jul. 17, 2017, PCT No. PCT/EP2017/068051
§ 371(c)(1), (2) Date Feb. 28, 2019,
PCT Pub. No. WO2018/054571, PCT Pub. Date Mar. 29, 2018.
Claims priority of application No. 16189976 (EP), filed on Sep. 21, 2016.
Prior Publication US 2019/0203242 A1, Jul. 4, 2019
Int. Cl. C12P 19/34 (2006.01); C12N 15/10 (2006.01); C12N 15/73 (2006.01); C12Q 1/6876 (2018.01)
CPC C12P 19/34 (2013.01) [C12N 15/10 (2013.01); C12N 15/73 (2013.01); C12Q 1/6876 (2013.01); C12N 2795/14042 (2013.01)] 16 Claims
 
1. A method for the recombinant production of DNA single strand molecules, comprising the steps of:
(1) providing a pseudogene nucleic acid,
wherein said pseudogene nucleic acid is a nucleic acid that comprises (i) at least one target DNA oligo- or polynucleotide sequence and (ii) at least one cleavage set of two self-cleaving DNA sequences flanking each target DNA oligo- or polynucleotide sequence, wherein said pseudogene nucleic acid comprises one cleavage set per the one target DNA oligo- or polynucleotide sequence, and two self-cleaving DNA sequences are linked in inverted order to each other to form the one cleavage set;
(2) integrating the pseudogene nucleic acid into a vector;
(3) transforming bacterial cells in a bacterial culture with said vector
(4) producing a precursor ssDNA from said vector under bacterial culture conditions, wherein said precursor ssDNA comprises the pseudogene nucleic acid;
(5) isolating the precursor ssDNA from the bacterial culture;
(6) digesting the precursor ssDNA under reaction conditions where the self-cleaving DNA sequences become active and cut off the at least one cleavage set;
(7) separating the at least one target single stranded DNA oligo- or polynucleotide sequence; and
(8) obtaining the at least one target single stranded DNA oligo- or polynucleotide sequence.