Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
The invention relates to a new method of sequencing a double stranded target polynucleotide. The two strands of the double stranded target polynucleotide are linked by a bridging moiety. The two strands of the target polynucleotide are separated using a polynucleotide binding protein and the target polynucleotide is sequenced using a transmembrane pore.
1. A method of sequencing a double stranded target polynucleotide, comprising:
(a) providing a double-stranded nucleic acid fragment, wherein the double-stranded nucleic acid fragment comprises a sense strand and an antisense strand;
(b) ligating a first adapter to the double-stranded nucleic acid fragment to form a first ligation product, wherein the first adaptor comprises a sense strand and an antisense strand, wherein the antisense strand of the first adaptor has a 3′ hairpin which can act as an intramolecular primer and wherein the first ligation product comprises the 3′ hairpin at its 3′ end after ligation;
(c) contacting the first ligation product to a polymerase, wherein the polymerase binds to the 3′ hairpin of the first ligation product; and
(d) incubating the polymerase-contacted first ligation product under conditions under which the polymerase extends from the antisense strand of the first adapter in a template-dependent nucleic acid synthesis reaction to produce a nucleic acid synthesis product; and
(e) ligating a second adapter to the nucleic acid synthesis product, wherein the second adapter comprises a hairpin loop bridging moiety.
2. The method of claim 1 , further comprising:
(f) sequencing the double stranded target polynucleotide using a single molecule sequencing technology.
3. The method of claim 1 , wherein the double stranded nucleic acid fragment is genomic DNA.
4. The method of claim 1 , wherein the first adaptor is a synthetic Y-adaptor.
5. The method of claim 1 , wherein the sense strand of the adaptor comprises a leader sequence.
6. The method of claim 1 , wherein the polymerase is a strand displacing polymerase.
7. The method of claim 6 , wherein the strand displacing polymerase is Phi29 or Klenow.
8. The method of claim 1 , wherein the first adaptor and the second adaptor each comprises mismatched recognition sites for a restriction endonuclease.