Methods using pores
View Patent ↗The invention relates to a method of identifying an individual nucleotide, comprising (a) contacting the nucleotide with a transmembrane protein pore so that the nucleotide interacts with the pore and (b) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide. The invention also relates to a method of sequencing nucleic acid sequences and kits related thereto.
1. A method of distinguishing an individual monophosphate nucleotide, comprising:
(a) contacting the monophosphate nucleotide with a transmembrane protein pore so that the monophosphate nucleotide interacts with the pore, wherein the pore comprises a molecular adaptor that facilitates the interaction between the monophosphate nucleotide and the pore, wherein the molecular adaptor is a cyclodextrin; and
(b) measuring a current passing through the pore during the interaction and thereby distinguishing the monophosphate nucleotide from other monophosphate nucleotides.
2. A method according to claim 1 , wherein the interaction involves the monophosphate nucleotide reversibly binding to the channel of the pore.
3. A method according to claim 1 , wherein the pore is α-hemolysin as shown in SEQ ID NO: 2 or a variant thereof.
4. A method according to claim 3 , wherein the variant is (M113R) 7 .
5. A method according to claim 1 , wherein the molecular adaptor is a heptakis-6-amino-β-cyclodextrin (am 7 -β-CD).
6. A method according to claim 1 , wherein the individual monophosphate nucleotide is a monophosphate ribonucleotide.
7. A method according to claim 6 , further comprising before step (a) digesting a ribonucleic acid (RNA) sequence to provide the individual monophosphate nucleotide.
8. A method according to claim 1 , wherein the individual monophosphate nucleotide is a monophosphate deoxyribonucleotide.
9. A method according to claim 8 , further comprising before step (a) digesting a deoxyribonucleic acid (DNA) sequence to provide the individual monophosphate nucleotide.
10. A method according to claim 7 , wherein more than one of the individual monophosphate nucleotides of the RNA sequence are contacted with the pore in a sequential manner such that the identity of the whole or part of the sequence may be determined.
11. A method of sequencing a target nucleic acid sequence, comprising:
(a) digesting an individual monophosphate nucleotide from one end of the target sequence using a processive exonuclease;
(b) contacting the monophosphate nucleotide with a transmembrane protein pore so that the monophosphate nucleotide interacts with the pore, wherein the pore comprises a molecular adaptor that facilitates the interaction between the monophosphate nucleotide and the pore, wherein the molecular adaptor is a cyclodextrin;
(c) measuring a current passing through the pore during the interaction and thereby determining the identity of the individual monophosphate nucleotide; and
(d) repeating steps (a) to (c) at the same end of the target sequence and thereby determining the sequence of the nucleic acid.
12. A method according to claim 9 , wherein more than one of the individual monophosphate nucleotides of the DNA sequence are contacted with the pore in a sequential manner such that the identity of the whole or part of the sequence may be determined.