US 12,071,650 B2
Method for producing double stranded polynucleotides based on oligonucleotides with selected and different melting temperatures
Joseph Brennan, Essex (GB); Daniel Bygrave, Essex (GB); Sangeeta Aditya, Essex (GB); and Raquel Sanches-Kuiper, Essex (GB)
Assigned to EVONETIX LTD, (GB)
Appl. No. 16/493,585
Filed by EVONETIX LTD, Cambridge (GB)
PCT Filed Mar. 13, 2018, PCT No. PCT/GB2018/050634
§ 371(c)(1), (2) Date Sep. 12, 2019,
PCT Pub. No. WO2018/167475, PCT Pub. Date Sep. 20, 2018.
Claims priority of application No. 17160666 (GB), filed on Mar. 13, 2017.
Prior Publication US 2021/0139957 A1, May 13, 2021
Int. Cl. C12Q 1/6811 (2018.01)
CPC C12Q 1/6811 (2013.01) 15 Claims
OG exemplary drawing
 
1. A method for producing a double-stranded polynucleotide, wherein the method comprises the steps:
(a) selecting and providing a group of single-stranded oligonucleotides for self-assembly into the double-stranded polynucleotide, the group comprising a plurality of overlapping complementary oligonucleotides, wherein each overlap between complementary oligonucleotides is selected to have a melting temperature (Tm) that differs from the melting temperatures of all other overlapping complementary oligonucleotides in the group, and wherein each overlap is selected to provide a difference between its Tm and the closest higher and lower Tm of greater than or equal to 2° C. as determined by nearest-neighbour model; and
(b) producing the double-stranded polynucleotide by self-assembly of the group of single-stranded oligonucleotides.