Enzyme stalling method
The invention relates to new methods of moving helicases past spacers on polynucleotides and controlling the loading of helicases on polynucleotides. The invention also relates to new methods of characterising target polynucleotides using helicases.
1. A single molecule sequencing method for characterising a template deoxyribonucleic acid (DNA) strand comprising:
(a) incubating the template DNA strand and a nucleotide handling protein bound thereto, with all components necessary to facilitate movement of the nucleotide handling protein along the template DNA strand, for a period of time wherein the movement of the nucleotide handling protein along the template DNA strand is not detected, wherein all components necessary to facilitate movement comprise dTTP; and
(b) initiating detection of the movement of the nucleotide handling protein along the template DNA strand; and
(c) determining one or more characteristics of a portion of the template DNA strand based on the detection of the movement of the nucleotide handling protein along the template DNA strand.
2. The method according to claim 1 , further comprising the step of fragmenting the template DNA strand prior to incubating step (a).
3. The method according to claim 1 , wherein the template DNA strand is provided as a fragmented strand.
4. The method according to claim 1 , wherein the template DNA strand is a fragment of 5 to 10 kb.
5. The method according to claim 1 , further comprising the step of ligating a single stranded DNA adaptor to the template DNA strand prior to incubating step (a).
6. The method according to claim 1 , further comprising the step of ligating a hairpin adaptor to the template DNA strand prior to incubating step (a).
7. The method according to claim 1 , further comprising the step of ligating a single stranded DNA adaptor to the template DNA strand and then further loading the nucleotide handling protein at a loading site of the adaptor, prior to incubating step (a).
8. The method according to claim 1 , further comprising the step of ligating a hairpin adaptor to the template DNA strand and then further loading the nucleotide handling protein at a loading site of the hairpin adaptor, prior to incubating step (a).
9. The method according to claim 1 , further comprising the step of ligating a single stranded DNA adaptor to the template DNA strand, wherein the nucleotide handling protein is already bound to the adaptor at a loading site of the adaptor, prior to incubating step (a).
10. The method according to claim 1 , further comprising the step of ligating a hairpin adaptor to the template DNA strand, wherein the nucleotide handling protein is already bound to the hairpin adaptor at a loading site of the adaptor, prior to incubating step (a).
11. The method according to claim 1 , further comprising the step of fragmenting the template DNA and ligating a single stranded DNA adaptor to the template DNA strand prior to incubating step (a).
12. The method according to claim 1 , further comprising the step of fragmenting the template polynucleotide, ligating a single stranded DNA adaptor to the template DNA strand and then loading the nucleotide handling protein at a loading site of the adaptor prior to incubating step (a).
13. The method according to claim 1 , further comprising the step of fragmenting the template DNA and ligating a single stranded DNA adaptor to the template DNA strand prior to incubating step (a), wherein the nucleotide handling protein is already bound to the loading site of the adaptor.
14. The method according to claim 1 , further comprising the step of fragmenting the template DNA and ligating a hairpin adaptor to the template DNA strand and then loading the nucleotide handling protein at a loading site of the hairpin adaptor prior to incubating step (a).
15. The method according to claim 1 , further comprising the step of fragmenting the template DNA and ligating a hairpin adaptor to the template DNA strand prior to incubating step (a), wherein the nucleotide handling protein is already bound to the loading site of the hairpin adaptor.
16. The method according to claim 1 , wherein the template DNA is ligated to a single stranded DNA adaptor comprising a pre-bound nucleotide handling protein.
17. The method according to claim 1 , wherein the template DNA is ligated to a hairpin adaptor comprising a pre-bound nucleotide handling protein.
18. The method according to claim 1 , wherein the template DNA is coupled to a surface.
19. The method according to claim 1 , wherein the components necessary to facilitate movement of the nucleotide handling protein along the template DNA strand comprise Mg 2+ and dTTP.
20. The method according to claim 1 , wherein detection of the movement of the nucleotide handling protein along the template DNA strand comprises detection of electrical and/or optical signals.
21. The method according to claim 1 , wherein the one or more characteristics are selected from the group consisting of: (i) the length of the template DNA strand, (ii) the identity of the template DNA strand, (iii) the sequence of the template DNA strand, (iv) the secondary structure of the template DNA strand and (v) whether or not the template DNA strand is modified.