Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
The claimed invention relates to a method of processing a polynucleotide, by obtaining a sense polynucleotide strand comprising a homopolymeric region that is longer that the reading section of a nanopore; synthesizing an antisense polynucleotide strand under conditions in which a nucleotide analog is incorporated at random in a reverse complement of the homopolymer region, such that the length of the homopolymer region in the antisense polynucleotide strand is shorter than the reading section of the nanopore; and moving the antisense polynucleotide strand through the nanopore such that a proportion of the antisense polynucleotide strand interacts with the nanopore.
1. A method of processing a polynucleotide, the method comprising:
a) obtaining a sense polynucleotide strand comprising a homopolymeric region than is longer that the reading section of a nanopore;
b) synthesizing an antisense polynucleotide strand under conditions in which a nucleotide analog is incorporated at random in a reverse complement of the homopolymer region, such that the length of the homopolymer region in the antisense polynucleotide strand is shorter than the reading section of the nanopore; and
(c) moving the antisense polynucleotide strand through the nanopore such that a portion of the antisense polynucleotide strand interacts with the nanopore.
2. The method of claim 1 , wherein the sense polynucleotide strand comprises
i) a sequence of nucleotides, wherein each nucleotide in the sequence is selected from a set of nucleotides; and
ii) the homopolymeric region, wherein the homopolymeric region comprising repeated instances of a member of the set of nucleotides, and
wherein step b) comprises combining a polymerase with the same set of nucleotides of step a) and a nucleotide analog, and maintaining the combination under conditions in which the nucleotide analog is incorporated at random in the reverse complement of the homopolymer region.
3. The method of claim 2 further comprising controlling the insertion rate of the nucleotide analog by varying the concentration of the nucleotide analog relative to the set of nucleotides.
4. The method of claim 1 , wherein the sense polynucleotide strand is connected to a single stranded leader.
5. The method of claim 4 , wherein the sense polynucleotide is connected to the single stranded leader via a stretch of abasic nucleotides.
6. The method of claim 1 , wherein the sense polynucleotide strand, in step a), comprises a hairpin loop having a free 3′ end.
7. The method of claim 6 , wherein the antisense polynucleotide strand is synthesized using a polymerase reaction that is nucleated from the free 3′ end of the hairpin loop.
8. The method of claim 1 , further comprising:
d) measuring the current passing through the nanopore during each interaction in step c) and thereby determining the sequence of the antisense polynucleotide strand.
9. The method of claim 8 , further comprising:
e) moving the sense polynucleotide strand through the nanopore such that a proportion of the sense polynucleotide strand interacts with the pore; and
f) measuring the current passing through the nanopore during each interaction in step e) thereby determining the sequence of the antisense polynucleotide strand.
10. The method of claim 9 , further comprising:
g) combining information from steps d) and f) to determine the length of the homopolymeric region.