DNA sequencing method
The present invention pertains to a method for determining the sequence of a polynucleotide, the method relying on the detection of a conformational change in an enzyme that interacts with and processes along the polynucleotide. The detection of a conformational change may be carried out by measuring changes in a fluorophore bound to the enzyme.
1 . A method for determining the sequence of a polynucleotide, comprising the steps of:
(i) reacting a target polynucleotide with an enzyme that is capable of interacting with and processing along the polynucleotide, under conditions sufficient to induce enzyme activity; and
(ii) detecting conformational changes in the enzyme as the enzyme processes along the polynucleotide.
2 . The method according to claim 1 , wherein the enzyme is a polymerase enzyme.
3 . The method according to claim 1 , wherein the enzyme is a helicase enzyme or a primase enzyme.
4 . The method according to claim 1 , wherein the enzyme is immobilised on a solid support.
5 . The method according to claim 4 , comprising a plurality of enzymes immobilised on the solid support.
6 . The method according to claim 1 , wherein the enzyme comprises a first bound detectable label, the characteristics of which alter as the enzyme undergoes a conformational change.
7 . The method according to claim 6 , wherein the enzyme comprises a second bound detectable label capable of interacting with the first label, wherein the degree of interaction is dependent on a conformational change in the enzyme.
8 . The method according to claim 6 , wherein a second detectable label is bound to a nucleotide brought into contact with the enzyme.
9 . The method according to claim 7 , wherein the first label is an energy acceptor and the second label is an energy donor, or wherein the first label is an energy donor and the second label is an energy acceptor, and wherein step (ii) is carried out by measuring energy transfer between the two labels.
10 . The method according to claim 8 , wherein the first label is an energy acceptor and the second label is an energy donor, or wherein the first label is an energy donor and the second label is an energy acceptor, and wherein step (ii) is carried out by measuring energy transfer between the two labels.
11 . The method according to claim 1 , wherein step (ii) is carried out using confocal microscopy.
12 . The method according to claim 11 , wherein step (ii) is carried out by fluorescence imaging.
13 . The method according to claim 6 , wherein step (ii) is carried out by measuring a polarisation effect consequent on the altered characteristics of the first label.
14 . The method according to claim 13 , wherein step (ii) is carried out by fluorescence polarisation anisotrophy.
15 . A method for determining the sequence of a polynucleotide, comprising detecting via fluorescence resonance energy transfer a conformational change in an enzyme that interacts with and processes along a target polynucleotide, thereby permitting determining the sequence of the polynucleotide.
16 . The method according to claim 15 , wherein the enzyme is a polymerase enzyme.
17 . The method according to claim 15 , wherein the enzyme is immobilised on a solid support.
18 . The method according to claim 16 , wherein the enzyme is immobilised on a solid support.
19 . A method for determining the sequence of a polynucleotide, comprising detecting a detectably-labelled enzyme that is capable of interacting with and processing along a target polynucleotide, wherein the label alters its detectable characteristics as the enzyme processes along the polynucleotide, thereby permitting determining the sequence of the polynucleotide.