Nanopore-based method and double stranded nucleic acid construct therefor
The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterised.
1. A double-stranded nucleic acid construct comprising:
(a) a double-stranded target polynucleotide construct; and
(b) a pair of hairpin loop-forming polynucleotides at both ends of the construct, wherein each pair comprises a 5′ hairpin loop-forming polynucleotide and a 3′ hairpin loop-forming polynucleotide,
wherein the 5′ hairpin loop-forming polynucleotide within a pair comprises a nucleotide sequence that is complementary to a nucleotide sequence of the 3′ hairpin loop-forming polynucleotide,
wherein hybridization of the 5′ hairpin loop-forming polynucleotide to the 3′ hairpin loop-forming polynucleotide produces a double-stranded portion, and
wherein the 3′ hairpin loop formed by the 3′ hairpin loop-forming polynucleotide has a higher melting temperature than the double-stranded portion.
2. The construct of claim 1 , wherein the 5′ hairpin loop-forming polynucleotide is formed from nucleotides consisting of deoxyadenosine, deoxythymidine, deoxyinosine, and deoxyzebularine.
3. The construct of claim 1 , wherein the 5′ hairpin loop-forming polynucleotide is formed from nucleotides consisting of deoxyadenosine, deoxythymidine, deoxyinosine, and deoxyzebularine; and the 3′ hairpin loop-forming polynucleotide is formed from nucleotides consisting of deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine.
4. The construct of claim 1 , wherein the nucleic acid construct is a deoxyribonucleic acid (DNA) construct.
5. The construct of claim 1 , wherein each hairpin loop-forming polynucleotide has a length of 1 to 110 nucleotides.
6. The construct of claim 1 , wherein each hairpin loop-forming polynucleotide has a length of about 5 to 110 nucleotides.
7. The construct of claim 1 , wherein the construct further comprises a spacer.
8. The construct of claim 7 , wherein the spacer is located on one or both sides of a hairpin loop-forming polynucleotide.
9. The construct of claim 7 , wherein the spacer is a iSpC3 group, iSp9 group or a iSp18 group.
10. The construct of claim 8 , wherein the spacer is a iSpC3 group, iSp9 group or a iSp18 group.
11. A method comprising contacting the construct of claim 1 with a polymerase.