Method for producing double stranded polynucleotides based on oligonucleotides with selected and different melting temperatures
AssigneeEvonetix Ltd
InventorsJoseph Brennan, Daniel Bygrave, Sangeeta Aditya, Raquel Sanches-Kuiper
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Abstract
A method for identifying a group of single-stranded oligonucleotides for self-assembly into a 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.
Claims15
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.
A method for providing multiple copies of a double-stranded polynucleotide comprising the steps:
- (a) selecting and providing a first set of oligonucleotides corresponding to one strand of the polynucleotide;
- and
- (b) selecting and providing a second set of oligonucleotides corresponding to the opposite strand of the polynucleotide, wherein each of the oligonucleotides of the second set of oligonucleotides overlaps with two oligonucleotides of the first set of oligonucleotides, except that two oligonucleotides at a 5′ or 3′ end of the double-stranded polynucleotide overlaps with only one complementary oligonucleotide, and 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 a group consisting of the first and second sets of oligonucleotides, 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
- (c) hybridising the first and second set of oligonucleotides.
Dependencies are inferred from the claim text.