NON-MASS DETERMINED BASE COMPOSITIONS FOR NUCLEIC ACID DETECTION

• US 20120183970A1
• Filed: 08/06/2010
• Published: 07/19/2012
• Est. Priority Date: 08/06/2009
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Sappenfield, Christopher C.
• Granted Patent: US 9,080,209 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

The present invention provides systems, methods, and compositions for nucleic acid detection based on non-mass determined base compositions. For example, in certain embodiments, base count data for a template nucleic acid is generated using an approach that does not measure molecular mass of the template nucleic acid (e.g., by sequencing the template nucleic acid) and a database comprising base count entries is queried to identify the target nucleic acid. In particular embodiments, sequencing is employed which is conducted in substantially real-time.

Claims

1. A method of identifying a template nucleic acid, the method comprising:

(a) determining at least a partial base count of at least a subsequence of at least one template nucleic acid and/or a complement thereof, using an approach that does not measure molecular mass of the template nucleic acid, to produce base count data; and

(b) querying a database comprising at least one base count entry corresponding to an identified nucleic acid to produce a match of the base count data with the base count entry, thereby identifying the template nucleic acid.

2. The method of claim 1, wherein the template nucleic acid comprises DNA or RNA.

3. The method of claim 1, wherein said determining is performed in substantially real-time.

4. The method of claim 1, wherein the template nucleic acid comprises a mammalian nucleic acid, a bacterial nucleic acid, a viral nucleic acid, a fungal nucleic acid, or a protozoal nucleic acid.

5. The method of claim 1, wherein the template nucleic acid is attached to a solid support.

6. The method of claim 1, comprising amplifying the template nucleic acid prior to or during (a).

7. The method of claim 1, comprising obtaining the template nucleic acid from one or more sample sources selected from the group consisting of:

an environmental sample and a sample derived from a subject.

8. The method of claim 1, wherein (a) comprises contacting the template nucleic acid with at least one nucleotide incorporating biocatalyst, labeled nucleotides, and at least one primer nucleic acid that is at least partially complementary to at least a subsequence of the template nucleic acid, under conditions whereby the nucleotide incorporating biocatalyst extends the primer nucleic acid to produce an extended primer nucleic acid by incorporating the labeled nucleotides at a terminal end of the extended primer nucleic acid, wherein nucleotides that comprise different nucleobases comprise different labels, wherein the different labels produce detectable signals as or after the labeled nucleotides are incorporated at the terminal end of the extended primer nucleic acid, which detectable signals identify the labeled nucleotides incorporated at the terminal end of the extended primer nucleic acid and/or complementary nucleotides in the template nucleic acid, and wherein the detectable signals are detected as or after the labeled nucleotides are incorporated at the terminal end of the extended primer nucleic acid to thereby determine the base count of the subsequence of the template nucleic acid and/or the complement thereof.

9. The method of claim 8, wherein labels comprise different fluorescent labels and wherein the detectable signals are detected using a fluorescence microscope.

10. The method of claim 8, wherein said at least one primer nucleic acid is a primer pair, wherein said primer pair is configured to hybridize with conserved regions of said two or more different bioagents and flank variable regions of said two or more different bioagents.

11. The method of claim 8, wherein the terminal end of the extended primer nucleic acid is the 3′

terminal end.

12. The method of claim 8, wherein the nucleotide incorporating biocatalyst comprises an enzyme selected from the group consisting of:

a polymerase, a terminal transferase, a reverse transcriptase, a polynucleotide phosphorylase, and a telomerase.

13. The method of claim 8, wherein the nucleotide incorporating biocatalyst comprises one or more modifications.

14. The method of claim 8, wherein the nucleotide incorporating biocatalyst is an enzyme derived from an organism that is selected from the group consisting of:

Thermus antranikianii, Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillus stearothermophilus.

15. The method of claim 8, wherein the nucleotide incorporating biocatalyst comprises a Φ

29 DNA polymerase.

16. The method of claim 8, wherein a label is attached to one of a heterocyclic base of a labeled nucleotide, a sugar moiety of a labeled nucleotide, and a phosphate group of a labeled nucleotide.

17. The method of claim 8, wherein a linker attaches a label to a labeled nucleotide.

18. The method of claim 8, wherein the extended primer nucleic acid is complementary to a subsequence of the template nucleic acid.

19. The method of claim 8, wherein the extended primer nucleic acid is complementary to a full-length sequence of the template nucleic acid.

20. The method of claim 8, wherein the primer nucleic acid comprises an intelligent primer.

21. The method of claim 8, wherein the label comprises a fluorescent dye, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme.

22. The method of claim 21, wherein the label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

23. The method of claim 21, wherein the label is a fluorescent dye selected from the group consisting of:

JOE, VIC, TET, HEX, PAM, R6G, R110, TAMRA, and ROX.

24. The method of claim 21, wherein the label is a radioisotope selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

25. The method of claim 1, comprising determining at least partial base counts of subsequences of two or more template nucleic acids and/or complements thereof, using an approach that does not measure molecular mass of the template nucleic acids to produce the base count data.

26. The method of claim 25, comprising querying the database to produce two or more matches of the base count data with two or more base count entries to thereby identify the template nucleic acid.

27. The method of claim 1, wherein identifying the template nucleic acid comprises identifying an organismal source of the template nucleic acid and/or one or more properties of the template nucleic acid.

28. The method of claim 27, wherein the properties of the template nucleic acid comprise a genotype, a virulence property, a pathogenic property, and/or a drug resistance property.

29. The method of claim 27, wherein said determining is performed in substantially real-time.

30. The method of claim 27, wherein the organismal source is identified at one or more taxonomic rank levels selected from the group consisting of:

a Domain, a Superphylum, a Superdivision, a Superclass, a Superorder, a Superfamily, a Superspecies, a Kingdom, a Phylum, a Division, a Class, a Legion, an Order, a Family, a Tribe, a Genus, a Species, a Subkingdom, a Subphylum, a Subclass, a Cohort, a Suborder, a Subfamily, a Subtribe, a Subgenus, a Subspecies, an Infrakingdom, a Branch, an Infraphylum, an Infraclass, an Infraorder, an Alliance, an Infraspecies, a Microphylum, a Pan/class, and a Parvorder.

31. A system, comprising:

at least one sequencing device;

at least one primer pair configured to hybridize with conserved regions of two or more different bioagents and flank variable regions of said two or more different bioagents; and

a computer readable medium having one or more logic instructions for directing a system to;

(a) determine at least a partial base count of at least a subsequence of at least one template nucleic acid and/or a complement thereof, using an approach that does not measure molecular mass of the template nucleic acid, to produce base count data; and

(b) query a database comprising at least one base count entry corresponding to an identified nucleic acid to produce a match of the base count data with the base count entry to thereby identifying the template nucleic acid.

32. The system of claim 31, wherein said sequencing device comprises a zero-mode waveguide.

33. The system of claim 32, further comprising one or more containers for packaging the zero-mode waveguide, or said primer pair.

34. A computer program product, comprising a computer readable medium having one or more logic instructions for directing a system to:

(a) determine at least a partial base count of at least a subsequence of at least one template nucleic acid and/or a complement thereof, using an approach that does not measure molecular mass of the template nucleic acid, to produce base count data; and

(b) query a database comprising at least one base count entry corresponding to an identified nucleic acid to produce a match of the base count data with the base count entry to thereby identifying the template nucleic acid.

35. A system, comprising:

a reaction vessel or substrate;

a detector configured to detect detectable signals produced in or on the reaction vessel or substrate, which detectable signals correspond to at least some nucleobases incorporated into a nucleic acid to generate nucleobase incorporation data;

a database of base count entries indexed to identified nucleic acids; and

a controller configured to correlate the nucleobase incorporation data with detected base counts and to query the database for a match between the detected base counts and the base count entries to thereby identify or detect the nucleic acids.

36. The system of claim 35, wherein the reaction vessel or substrate comprises at least one zero-mode waveguide.

37. The system of claim 35, wherein the detector comprises a fluorescence microscope.

38. The system of claim 35, comprising at least one material transfer component that transfers material to and/or from the reaction vessel or substrate.

39. The system of claim 35, comprising at least one thermal modulator configured to modulate temperature in the reaction vessel or substrate.

40. The system of claim 35, wherein the reaction vessel or substrate comprises at least one primer nucleic acid is selected from the primer nucleic acids disclosed in one or more of the published documents incorporated by reference herein.

41. The system of claim 35, comprising at least one nucleic acid sample preparation component and/or at least one nucleic acid amplification component.

42. A method of identifying a target nucleic acid in a sample comprising:

(a) at least partially sequencing said target nucleic acid, or amplified sequences thereof, to generate sequence data;

(b) determining at least a partial base count from said sequence data to generate base count data; and

(c) querying a database comprising at least one base count entry corresponding to said target nucleic acid to produce a match of said base count count data with said base count entry, thereby identifying said target nucleic acid.

43. The method of claim 42, wherein the molecular mass of said target sequence or said amplified sequences is not determined in said identifying said target nucleic acid.

44. The method of claim 42, wherein said sequencing is performed in real-time or substantially real-time.

45. The method of claim 44, wherein said identifying said target nucleic acid is performed in substantially real-time.

46. The method of claim 42, wherein said at least partially sequencing said target nucleic acid is performed with a device comprising at least one zero-mode waveguide.

47. A method of identifying a target nucleic acid in a sample comprising:

(a) amplifying one or more segments of said nucleic acid in said sample using at least one purified oligonucleotide primer pair to generate amplified sequences, wherein said primer pair is configured to hybridize with conserved regions of two or more different bioagents and flank variable regions of said two or more different bioagents;

(b) at least partially sequencing said amplified sequences to generate sequence data;

(c) determining at least a partial base count from said sequence data to generate base count data; and

(d) querying a database comprising at least one base count entry corresponding to said target nucleic acid to produce a match of said base count count data with said base count entry, thereby identifying said target nucleic acid.

48. The method of claim 47, wherein the molecular mass of said target sequence or said amplified sequences is not determined in said identifying said target nucleic acid.

49. The method of claim 47, wherein said sequencing is performed in real-time or substantially real-time.

50. The method of claim 49, wherein said identifying said target nucleic acid is performed in substantially real-time.

51. The method of claim 49, wherein said at least partially sequencing said target nucleic acid is performed with a device comprising at least one zero-mode waveguide.

52. The method of claim 47, wherein said primer pair comprises forward and reverse primers that are about 20 to 35 nucleobases in length.

53. A system, comprising:

(a) a sequencing device configured to generate nucleic acid sequence data corresponding to the nucleic acid sequence of one or more amplicons produced using at least one purified oligonucleotide primer pair that comprises forward and reverse primers, wherein said primer pair comprises nucleic acid sequences that are substantially complementary to nucleic acid sequences of two or more different bioagents; and

(b) a controller operably connected to said sequencing device, said controller configured to;

i) determine at least a partial base count from said nucleic acid sequence data to generate base count data, and

ii) query a database with said base count data, wherein said database comprises at least one base count entry corresponding to a target nucleic acid.