Method and apparatus for DNA sequencing using a local sensitive force detector

• US 6,280,939 B1
• Filed: 09/01/1998
• Issued: 08/28/2001
• Est. Priority Date: 09/01/1998
• Status: Expired due to Fees

• Current Assignee: Bruker Nano, Inc. (Bruker Corporation)
• Original Assignee: Veeco Instruments Incorporated
• Inventors: Allen, Michael J.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/145,352
• Time in Patent Office: 1,092 Days
• Field of Search: 435/6, 435/91.1, 536/25.3
• US Class Current: 435/6.19
• CPC Class Codes: B82Y 35/00 Methods or apparatus for measurement or analysis of nanostructures
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: G01Q 60/42 Functionalisation
• CPC Class Codes: Y10S 977/853 Biological sample
• CPC Class Codes: Y10S 977/959 Of disease state

Abstract

DNA sequencing is performed in real time using an atomic force microscope (AFM). The AFM'"'"'s probe detects motions that occur when a polymerase incorporates nucleotides into a growing polynucleotide chain and a newly formed double-stranded polynucleotide helix translocates (or “ratchets”) through the polymerase'"'"'s reaction site. These motions generate a mechanical force that is reflected, either directly or indirectly, by motion of the AFM cantilever. The resulting changes in cantilever motion are detected and can be recorded as an indication that a nucleotide has been incorporated into the DNA template. To determine which nucleotide type has been incorporated, a characteristic of the incorporation of at least one nucleotide type of interest is flagged so as to be distinguishable from the corresponding characteristics of the incorporation of nucleotides of other types. In a preferred embodiment, the average incorporation delay for nucleotides of the nucleotide type of interest are flagged such that the average incorporation delay for nucleotides of the nucleotide type of interest is unique when compared to the average incorporation delay of nucleotides of other nucleotide types.

Claims

1. A method comprising:

(A) incorporating nucleotides of more than one nucleotide type into a growing polynucleotide chain by a polymerase during a polymerase reaction in which the order of incorporation is driven by base pair rules and a delay occurs between each successive nucleotide incorporation;

(B) controlling at least one characteristic of the incorporation of at least one nucleotide type of interest into the growing polynucleotide chain such that the concentration of a nucleotide of interest is different from the concentration of at least one other nucleotide and wherein the incorporation delay of the nucleotide of interest is different from the average incorporation delay for nucleotides of at least one other nucleotide type;

(C) locating the polymerase with a local sensitive force detector;

(D) detecting by using the local sensitive force detector, a motion associated with the nucleotide incorporation into the growing polynucleotide chain; and

(E) producing data using a computer to transform detected incorporation data into nucleofide data.

2. A method as defined in claim 1, wherein the locating step comprises,

(A) operating the detector in a scanning mode to locate a polynucleotide/polymerase complex, and then

(B) switching from the scanning mode to a data acquisition mode in which a probe tip of the detector is placed at the polynucleotide/polyinerase complex.

3. A method as defined in claim 1, wherein the locating step comprises operating the detector in an approaching mode to locate a polynucleotide/polymerase complex,

and then

if a complex is found, then switching from the approaching mode to a data acquisition mode in which a probe tip of the detector is placed at the polynucleotide/polymerase complex, or

if a complex is not found, then switching from the approaching mode to a scanning mode to locate a polynucleotide/polymerase complex.

4. A method as defined in claim 1, wherein the locating step comprises operating the detector in an approaching mode to locate a polynucleotide/polymerase complex,

and then

if a complex is found, then switching from the approaching mode to a data acquisition mode in which a probe tip of the detector is placed at the polynucleotide/polynerase complex, or

if a complex is not found, then re-engaging to locate a polynucleotide/polymerase complex.

5. A method as defined in claim 1, wherein the detecting step comprises using a probe of the detector to detect a motion occurring when a nucleotide is incorporated into the growing polynucleotide chain and a newly formed structure translocates through a reaction site of the polynerase.

6. A method as defined in claim 5, wherein the detector comprises an atomic force microscope (AFM) and wherein the motion generates a mechanical force that is reflected by a motion of a cantilever of the AFM.

7. A method as defined in claim 1, wherein, during the incorporating step, incorporation delays occur between successive nucleotide incorporations, and wherein the controlling step comprises controlling an average incorporation delay for nucleotides of the nucleotide type of interest to differ from an average incorporation delay for nucleotides of at least one other nucleotide type.

8. A method as defined in claim 7, wherein the controlling step comprises controlling the average incorporation delays for nucleotides of all nucleotide types other than the nucleotide type of interest to be at least substantially the same as one another.

9. A method as defined in claim 8, further comprising controlling an average incorporation delay for nucleotides of at least two nucleotide types of interest to differ from an average incorporation delay for nucleotides of at least one other nucleotide type.

10. A method as defined in claim 1, wherein the controlling step comprises controlling an amplitude of a motion as measured by a probe detector occurring when a nucleotide is incorporated into the growing polynucleotide chain and a newly formed structure translocates through a reaction site of the polymerase.

11. A method as defined in claim 1, wherein the controlling step comprises conjugating a nucleotide of the nucleotide type of interest with a molecule.

12. A method as defined in claim 1, wherein the controlling step comprises:

(A) performing a plurality of polymerization reactions, and

(B) during each of the polymerization reactions, controlling the at least one characteristic of the incorporation of a different nucleotide type of interest such that the incorporation of a nucleotide of the nucleotide type of interest is distinguishable from the incorporation of nucleotides of all other nucleotide types,

and further comprising determining, based on the controlling and detecting steps, which nucleotide type was incorporated into the growing polynucleotide chain during the incorporating step.

13. A method as defined in claim 12, wherein the determining step comprises:

(A) examining each of the reactions separately, and, for each of the reactions, (1) detecting whether or not a nucleotide of the nucleotide type of interest for that reaction is incorporated and (2) generating a set of data, and

(B) adding a plurality of sets of data together to generate nucleotide sequence data for all of the data sets.

14. A method as defined in claim 1, wherein, during the controlling step, at least one characteristic of the incorporation for the nucleotide type of interest is unique when compared to the corresponding characteristics of the incorporation for all other nucleotide types in the polymerization reaction.

15. A method as defined in claim 14, further comprising determining, based on the controlling and detecting steps, which nucleotide type was incorporated into the growing polynucleotide chain during the detecting step,

wherein the polymerization reaction is performed in a single reaction and a plurality of nucleotide types each have a unique incorporation characteristic when compared to corresponding incorporation characteristics of other nucleotide types,

wherein the detecting step comprises detecting the unique incorporation characteristic of each of the nucleotides types, and

wherein the determining step comprises determining, from the unique incorporation characteristic, which nucleotide type was incorporated into each location of interest of the growing polynucleotide chain.

16. A method as defined in claim 1, wherein the detecting steps further comprises subtracting background noise from detected probe operating parameters, the background noise arising from at least one of unintended probe motion and enzyme motion.

17. A method as defined in claim 1, wherein the incorporating step comprises controlling the polymerization reaction such that a rate of the polymerization reaction permits detection of incorporations.

18. A method as defined in claim 1, wherein the incorporating step comprises controlling the polymerization reaction such that a rate of the polymerization reaction is outside of a range in which natural variations in average incorporation delays would obscure acquired data.

19. A method as defined in claim 1, wherein the incorporating step comprises using a polymerase that is attached to a substrate.

20. A method as defined in claim 1, wherein the incorporating step comprises starting the incorporation at the beginning of the growing polynucleotide chain.

21. A method as defined in claim 1, further comprising repeating the incorporating, controlling, and detecting steps for a plurality of polymerase reactions.

22. A method as defined in claim 1, wherein the incorporating step comprises controlling the temperature of the polymerase reaction.

23. A method as defined in claim 1, further comprising detecting mutations during the polymerase reaction by using the method to determine which nucleotide type was incorporated into the growing polynucleotide chain for a small number of nucleotides.

24. A method as defined in claim 1, wherein the detector comprises an atomic force microscope (AFM).

25. A method as defined in claim 1, wherein the detecting step comprises detecting a motion of the polymerase.

26. A method comprising:

(A) incorporating nucleotides of more than one nucleotide type into a growing polynucleotide chain by a polymerase during a polymerase reaction in which the order of incorporation is driven by base pair rules;

(B) controlling at least one characteristic of the incorporation of at least one nucleotide type of interest into the growing polynucleotide chain such that the incorporation of nucleotides of the nucleotide type of interest is distinguishable from the incorporation of nucleotides of at least one other nucleotide type;

(C) locating the polymerase with a local sensitive force detector;

(D) detecting using the local sensitive force detector, a motion associated with the nucleotide incorporation into the growing polynucleotide chain;

(E) producing data using a computer to transform detected incorporation data into nucleotide data; and

(F) withholding at least one reaction component until a time just prior to a time at which data acquisition begins.

27. A method comprising:

(A) incorporating nucleotides of more than one nucleotide type into a growing polynucleotide chain by a polymerase during a polyrnerase reaction in which the order of incorporation is driven by base pair rules;

(B) controlling at least one characteristic of the incorporation of at least one nucleotide type of interest into the growing polynucleotide chain such that the incorporation of nucleotides of the nucleotide type of interest is distinguishable from the incorporation of nucleotides of at least one other nucleotide type;

(C) locating the polymerase with a local sensitive force detector in Tapping Mode;

(D) detecting using the local sensitive force detector, a motion associated with the nucleotide incorporation into the growing polynucleotide chain; and

(E) producing data using a computer to transform detected incorporation data into nucleotide data.

28. A method comprising:

(A) incorporating nucleotides of more than one nucleotide type into a growing polynucleotide chain by a polymerase during a polymerase reaction in which the order of incorporation is driven by base pair rules;

(B) controlling at least one characteristic of the incorporation of at least one nucleotide type of interest into the growing polynucleotide chain such that the incorporation of nucleotides of the nucleotide type of interest is distinguishable from the incorporation of nucleotides of at least one other nucleotide type and wherein secondary structure of a polynucleotide template formed by the incorporation is minimized;

(C) locating the polymerase with a local sensitive force detector;

(D) detecting using the local sensitive force detector, a motion associated with the nucleotide incorporation into the growing polynucleotide chain; and

(E) producing data using a computer to transform detected incorporation data into nucleotide data.