MOLECULAR DIAGNOSTICS REAGENTS AND METHODS

• US 20100330575A1
• Filed: 06/29/2010
• Published: 12/30/2010
• Est. Priority Date: 12/23/2004
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Inventors: Miller, Cary James, Collier, Gordon Bruce, MacLeod, Jason Andrew, Dicke, William Charles, Wood, John Allister, Nemeth, Attila Csaba
• Granted Patent: US 8,048,633 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502769 characterised by multiphase flow arrangements
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/0332 using permanent magnets
• CPC Class Codes: B03C 1/0335 using coils
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]

Abstract

The present invention relates to automated devices and methods for the extraction of nucleic acids from cells, the amplification of segments of nucleic acid and the detection of nucleic acids, all in a convenient and portable manner. The invention is particularly suited for use in point-of-care medical diagnostics testing.

Claims

1. A self-annealing oligonucleotide primer for use in a nucleic acid amplification assay, comprising:

a primer region;

a polymerase blocking region; and

a single stranded hybridization region, said primer predominantly having a secondary structure in solution in a first temperature range and not in a second, higher temperature range,

said primer capable of binding a target nucleic acid in said second temperature range and not in said first temperature range,

wherein said secondary structure involves one or more self-complimentary regions between portions of said primer region and portions of said single stranded hybridization region which hybridize, in at least one of an intra-molecular and an inter-molecular manner, in said first temperature range and not in said second temperature range.

2. A primer of claim 1, with an attached detectable label.

32. A primer of claim 1, wherein said target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.

33. A primer of claim 1, wherein said target nucleic acid is extracted from blood, a buccal swab, tissue, a bodily fluid, an environmental sample, a surface of a material, a plant, an animal, a bacteria and a fungi.

34. A primer of claim 1, wherein said primer is selected from the group deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid and hexa-polyethylene glycol modified nucleic acid.

35. A primer of claim 1, wherein said polymerase blocking region is a phosphoramidite.

36. A primer of claim 1, wherein said polymerase blocking region comprises 18-O-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

37. A primer of claim 1, wherein said single stranded hybridization region is selected from the group consisting of nucleic acids, ribonucleic acids, peptide nucleic acids, modified nucleic acids.

38. A primer of claim 2, wherein said detectable label is selected from the group consisting of biotin, streptavidin, FAM, DNP, cholesterol, and a DNA glycoconjugate.

39. A primer of claim 2, wherein said detectable label is biotin.

3. A method of performing a nucleic acid amplification assay comprising:

(a) combining reagents, DNA polymerase, a target nucleic acid, and a modified primer,

said modified primer comprising a sequence of bases complimentary to a first region of said target nucleic acid, a polymerase blocking region, a single stranded hybridization region attached to said polymerase blocking region, and a detectable label, said primer predominantly having a secondary structure in solution in a lower temperature range and not in an elevated temperature range, wherein said secondary structure involves annealing one or more self-complimentary regions between portions of said primer region and portions of said single stranded hybridization region,

wherein said modified primer is capable of priming said target nucleic acid and hybridizing with an oligonucleotide complimentary to said single stranded hybridization region at a first elevated temperature range, and substantially not in a second lower temperature range;

(b) cycling the mixture of (a) at said first temperature range to provide multiple copies of an amplicon incorporating said modified primer, wherein said incorporated primer is capable of hybridizing with an oligonucleotide complimentary to said single stranded hybridization region in said first elevated temperature range and in said second lower temperature range;

(c) reducing the temperature of the mixture to the second temperature range;

(d) exposing said mixture to a capture oligonucleotide complimentary to said single stranded hybridization region;

(e) hybridizing said single stranded hybridization region of said amplicon incorporating said modified primer, with said capture oligonucleotide; and

(f) detecting said label associated with said hybridization.

5. The method of claims 3 and 4, wherein said target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid and modifications and derivatives thereof.

6. The method of claims 3 and 4, wherein said target nucleic acid is extracted from blood, a buccal swab, tissue, a bodily fluid, an environmental sample, a surface of a material, a plant, an animal, a bacteria and a fungi.

8. The method of claims 3 and 4, wherein said DNA polymerase is Taq polymerase.

9. The method of claims 3 and 4, wherein said DNA polymerase is thermococcus kodakiensis polymerase.

10. The method of claims 3 and 4, wherein said primer is selected from the group deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid and hexa-polyethylene glycol modified nucleic acid.

11. The method of claims 3 and 4, wherein said polymerase blocking region is a phosphoramidite.

12. The method of claims 3 and 4, wherein said polymerase blocking region comprises 18-O-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

13. The method of claims 3 and 4, wherein said single stranded hybridization region is selected from the group consisting of nucleic acids, ribonucleic acids, peptide nucleic acids, modified nucleic acids.

14. The method of claims 3 and 4, wherein said sequence of bases complimentary to a first region has a reactive 3′

-hydroxyl.

15. The method of claims 3 and 4, wherein said detectable label is selected from the group consisting of biotin, streptavidin, FAM, DNP, cholesterol, and a DNA glycoconjugate.

16. The method of claims 3 and 4, wherein said detectable label is biotin.

17. The method of claims 3 and 4, wherein cycling is isothermal.

18. The method of claims 3 and 4, wherein cycling is between a first and second temperature.

19. The method of claims 3 and 4, wherein said capture oligonucleotide is immobilized on an optical surface.

20. The method of claims 3 and 4, wherein said capture oligonucleotide is immobilized on a bead.

21. The method of claims 3 and 4, wherein said capture oligonucleotide is immobilized on an electrode.

22. The method of claims 3 and 4, wherein the single stranded hybridization region of the modified primer hybridizes to the capture oligonucleotide.

23. The method of claims 3 and 4, wherein said detection is electrochemical.

24. The method of claims 3 and 4, wherein said detection is an optical method.

25. The method of claims 3 and 4, wherein said detection is colorimetric.

26. The method of claims 3 and 4, wherein said detection is fluorescent.

27. The method of claims 3 and 4, wherein said detection is enzymatic.

28. The method of claims 3 and 4, wherein said label is exposed to a second moiety which has a second label, said second moiety binding specifically to the first label, and wherein detection involves detection of said second label.

29. The method of claims 3 and 4, wherein said label is exposed to a second moiety which has a second label, said second moiety binding specifically to said first label, wherein detection involves detection of said second label, wherein said label is selected from the group alkaline phosphatase, glucose oxidase, sarcosine oxidase, cholesterol oxidase, uricase, L-amino acid oxidase, D-amino acid oxidase, urease, ascorbate oxidase, horseradish peroxidase and luciferase.

30. The method of claims 3 and 4, wherein said label is biotin and is exposed to a streptavidin-labeled alkaline phosphatase and detection of said label is based on detection of alkaline phosphatase activity.

31. The method of claims 3 and 4, wherein said label is biotin and is exposed to a streptavidin-labeled alkaline phosphatase and detection of biotin is based on detection of said alkaline phosphatase, and wherein alkaline phosphatase converts p-aminophenol phosphate to p-aminophenol and is detected electrochemically.

4. A method of performing a nucleic acid amplification assay comprising:

(a) combining reagents, DNA polymerase, a target nucleic acid, a modified primer and a second primer,

said modified primer comprising a sequence of bases complimentary to a first region of said target nucleic acid, a polymerase blocking region and a single stranded hybridization region attached to said polymerase blocking region, said primer predominantly having a secondary structure in solution in a lower temperature range and not in an elevated temperature range, wherein said secondary structure involves annealing one or more self-complimentary regions between portions of said primer region and portions of said single stranded hybridization region,

wherein said modified primer is capable of priming said target nucleic acid and hybridizing with an oligonucleotide complimentary to said single stranded hybridization region at a first elevated temperature range, and substantially not in a second lower temperature range,

said second primer comprising a sequence of bases complimentary to a second region of said target nucleic acid and having a detectable label;

(b) cycling the mixture of (a) at said first temperature range to provide multiple copies of an amplicon incorporating said modified primer and said second primer, wherein said incorporated modified primer is capable of hybridizing with an oligonucleotide complimentary to said single stranded hybridization region in said first elevated temperature range and in said second lower temperature range;

(c) reducing the temperature of the mixture to the second temperature range;

(d) exposing said mixture to a capture oligonucleotide complimentary to said single stranded hybridization region;

(e) hybridizing said single stranded hybridization region of said amplicon incorporating said modified primer, with said capture oligonucleotide; and

(f) detecting said label associated with said second primer incorporated into said amplicon.

7. The method of claim 4, wherein said reagents comprise reagents for a polymerase chain reaction amplification.