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 comprising:
(i) preparing a nucleic acid construct by ligating a polynucleotide bridging moiety to a 3′ terminus of a first strand and a 5′ terminus of a second strand of a double stranded nucleic acid, wherein the first strand of the double stranded nucleic acid comprises a first nucleic acid sequence and the second strand of the double stranded nucleic acid comprises a second nucleic acid sequence that is complementary to the first nucleic acid sequence;
(ii) using a single molecule sequencing apparatus to perform a sequencing reaction that reads through the first strand, the bridging moiety, and the second strand to produce orthogonal proof-reading sequence information for a methylated nucleobase in the first strand of the construct, wherein the movement of the nucleic acid construct through the single molecule sequencing apparatus is controlled by a Phi29 DNA polymerase;
(iii) comparing the sequence information between the first nucleic acid sequence and the second nucleic acid sequence; and
(iv) determining the presence of a methylated nucleobase in the first sequence.
2. The method of claim 1 , wherein the polynucleotide bridging moiety comprises a marker.
3. The method of claim 2 , wherein the marker is an abasic DNA or a sequence specific nucleic acids.
4. The method of claim 2 , wherein the method further comprising obtaining a signal from the marker in sequencing the bridging moiety that is indicative of the first strand having been sequenced.
5. The method of claim 1 , wherein the Phi29 DNA polymerase is not chemically modified.
6. The method of claim 1 , wherein the Phi29 DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 6.
7. The method of claim 1 , wherein the preparing comprises synthesizing a complement of the first sequence to produce the double stranded nucleic acid, wherein the complement contains nucleobases selected from adenine, guanine, thymine, uracil, and cytosine.
8. The method of claim 1 , wherein the first nucleic acid sequence is present in a biological sample, wherein the biological sample is obtained or extracted from an organism or a microorganism.
9. The method of claim 1 , wherein the bridging moiety is a hairpin loop.
10. The method of claim 9 , wherein the hairpin loop is 4-100 nucleotides in length.
11. The method of claim 1 , wherein the hairpin loop is 4-8 nucleotides in length.
12. The method of claim 1 , wherein the first nucleic acid sequence is a DNA sequence.
13. The method of claim 1 , wherein the methylated nucleobase is 5-methylcytosine, 5-hydroxymethylcytosine, or methylcytidine.
14. The method of claim 1 , wherein the double-stranded nucleic acid comprises hemi-genomic DNA.