Non-mass determined base compositions for nucleic acid detection

• US 9,080,209 B2
• Filed: 08/06/2010
• Issued: 07/14/2015
• Est. Priority Date: 08/06/2009
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Sappenfield, Christopher C.
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US13/389,139
• Publication Number:
  US 20120183970A1
• Time in Patent Office: 1,803 Days
• Field of Search: None
• US Class Current: 1/1
• 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 classifying a bioagent template nucleic acid according to a subgenus or species, said method comprising:

(a) amplifying a bioagent template nucleic acid in a sample using a primer pair to generate an amplicon,

wherein said primer pair hybridizes to said bioagent template nucleic acid and said amplicon at regions conserved among a genus of bioagents, and

wherein said conserved regions flank a variable region that differs between at least two or more species of said genus of bioagents;

(b) determining a partial base count of at least a subsequence of said variable region using a method that does not measure molecular mass of said template nucleic acid by mass spectrometry, to produce partial base count data, wherein said partial base count data provides the number but not the linear order of only one, two, or three nucleobase types, and wherein said subsequence of said variable region is at least 17 nucleobases in length; and

(c) querying a database in silico with said partial base count data,

wherein said database comprises a collection of partial base composition entries that are each indexed to both;

i) a particular subgenus or species, and ii) said primer pair used to generate said amplicon,

wherein each of said partial base count entries provides the number but not the linear order of only one, two or three nucleobase types, and

wherein said querying is performed by a computer processor coupled to a controller configured to query said database for a match of said partial base count data with at least one of said partial base count entries, thereby classifying said bioagent template nucleic acid according to said subgenus or said species.

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

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

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

6. The method of claim 1, wherein said subsequence of said variable region is at least 72 bases in length.

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

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

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

terminal end.

11. The method of claim 8, wherein said 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.

12. The method of claim 8, nucleotide incorporating biocatalyst comprises one or more modifications.

13. The method of claim 8, wherein said 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.

14. The method of claim 8, wherein said nucleotide incorporating biocatalyst comprises a Φ

29 DNA polymerase.

15. 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.

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

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

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

19. The method of claim 8, wherein said 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.

20. The method of claim 19, wherein said 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.

21. The method of claim 19, wherein said label is a fluorescent dye selected from the group consisting of:

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

22. The method of claim 19, wherein said label is a radioisotope selected from the group consisting of:

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

26. The method of claim 1, wherein said partial base count entry provides the number but not linear order of A nucleobases and G nucleobases.

27. The method of claim 1, wherein said partial base count entry provides the number but not linear order of A nucleobases, C nucleobases and T nucleobases.

28. The method of claim 1, wherein said partial base count entry provides the number but not linear order of G nucleobases, C nucleobases and T nucleobases.

29. The method of claim 1, wherein said partial base count entry provides the number but not linear order of C nucleobases and T nucleobases.

23. A method of classifying a bioagent target nucleic acid in a sample according to a subspecies comprising:

(a) amplifying a bioagent template nucleic acid in a sample using a primer pair to generate an amplicon,

wherein said primer pair hybridizes to said bioagent template nucleic acid and said amplicon at regions conserved among a species of bioagents, and

wherein said conserved regions flank a variable region that differs between at least two or more subspecies of said species of bioagents;

(b) determining a partial base count of at least a subsequence of said variable region using a method that does not employ mass spectrometry to produce partial base count data, wherein said partial base count data provides the number, but not the linear order of, only one, two, or three nucleobase types, and wherein said subsequence of said variable region is at least 17 nucleobases in length; and

(c) querying a database in silico with said partial base count data,

wherein said database comprises a collection of partial base composition entries that are each indexed to both;

i) a particular subspecies and ii) said primer pair used to generate said amplicon,

wherein each of said partial base count entries provides the number but not the linear order of only one, two or three nucleobase types, and

wherein said querying is performed by a computer processor coupled to a controller configured to query said database for a match of said partial base count data with at least one of said partial base count entries, thereby classifying said bioagent target nucleic acid according to said subspecies.

24. The method of claim 23, wherein said classifying said target nucleic acid is performed in real-time.

25. The method of claim 23, wherein said subsequence of said variable region is at least 72 bases in length.