1. A cigarette lighter with built-in clip for attachment of a chain or cord, which comprises:
a.) a unitary main housing having a top, a bottom and sidewalls, said bottom being predominantly flat so as to be standable upright on a horizontal surface, said main housing including lighter fuel storage means, conventional movement means for movement of lighter fuel to a flame area on said top, a flint striker located on said top for directing flint to said flame area and a control lever to permit and prevent fuel from flowing from said storage means to said flame area; and,
b.) a clip mechanism formed within a portion of said main housing and including a cut-out area to receive a chain or cord, a clip bar having a first position, being a closed position wherein a first face of said clip bar is substantially flush with said bottom of said main housing and having a second position, being an open position, said clip bar being rotatably connected to said main body so as to be moveable in an arc, a clip bar stop located on said main body, and a spring mechanism biasing said clip bar to its first position wherein said spring mechanism contacts said main body and a second face of said clip bar opposite said first face.
2. The cigarette lighter with built-in clip for attachment of a chain or cord, of claim 1 wherein said clip mechanism is located at said bottom of said main housing.
3. The cigarette lighter with built-in clip for attachment of a chain or cord, of claim 2 wherein said clip bar is flat and parallel to said main housing bottom.
4. The cigarette lighter with built-in clip for attachment of a chain or cord, of claim 1 wherein said main housing is made of a material selecting from the group consisting of metal, plastic and combination thereof.
5. The cigarette lighter with built-in clip for attachment of a chain or cord, of claim 4 wherein said clip mechanism is made of a material selected form the group consisting of metal, plastic and combination thereof.
6. A cigarette lighter with built-in clip for attachment of a chain or cord, which comprises:
a.) a unitary main housing having a top, a bottom and sidewalls, said bottom being predominantly flat so as to be standable upright on a horizontal surface, said main housing including lighter fuel storage means, conventional movement means for movement of lighter fuel to a flame area on said top, a flint striker located on said top for directing flint to said flame area and a control lever to permit and prevent fuel from flowing from said storage means to said flame area; and,
b.) a clip mechanism formed within a portion of said main housing and including a cut-out area formed in an arc shape to receive a chain or cord, a clip bar having a first position, being a closed position wherein a first face of said clip bar is substantially flush with said bottom of said main housing and having a second position, being an open position, said clip bar being rotatably connected to said main body so as to be moveable in an arc, a clip bar stop located on said main body, and a spring mechanism biasing said clip bar to its first position wherein said spring mechanism contacts said main body and a second face of said clip bar opposite said first face.
7. The cigarette lighter with built-in clip for attachment of a chain or cord, of claim 6 wherein said clip bar is flat and parallel to said main housing bottom.
8. The cigarette lighter with built-in clip for attachment of a chain or cord, of claim 6 wherein said main housing is made of a material selecting from the group consisting of metal, plastic and combination thereof.
9. The cigarette lighter with built-in clip for attachment of a chain or cord, of claim 6 wherein said clip mechanism is made of a material selected form the group consisting of metal, plastic and combination thereof.
10. The cigarette lighter with built-in clip for attachment of a chain or cord, of claim 6 wherein said main housing is made of plastic.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A composition for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, said composition comprising an upstream primer, downstream probe having a 3\u2032 flap and a 3\u2032-5\u2032 exonuclease.
2. The composition of claim 2, wherein said 3\u2032-5\u2032 exonuclease is a DNA polymerase.
3. The composition of claim 1, wherein the 3\u2032-5\u2032 exonuclease is thermostable.
4. The composition of claim 1, wherein a 5\u2032 region of the downstream probe is complementary to the target.
5. The composition of claim 1, wherein the downstream probe comprises at least one labeled moiety capable of providing a signal.
6. The composition of claim 1, wherein the downstream probe comprises an interactive pair of labels.
7. The composition of claim 6, wherein said interactive pair of labels comprises a quencher moiety and a fluorescent moiety.
8. The composition of claim 6, wherein at least one member of the interactive pair of labels is operatively coupled to the 3\u2032 flap of the downstream probe.
9. The composition of claim 5, wherein the at least one labeled moiety is operatively coupled to the 3\u2032 flap of the downstream probe.
10. The composition of claim 6, wherein a first member of the interactive pair of labels is operatively coupled to the 3\u2032 flap and a second member of the interactive pair of labels is operatively coupled to the 5\u2032 region of the downstream probe.
11. A labeled oligonucleotide pair, comprising:
a first oligonucleotide comprising a 5\u2032 region and a 3\u2032 region, wherein the 5\u2032 region is complementary to a target nucleic acid and the 3\u2032 region is non-complementary to the target nucleic acid, and wherein the 3\u2032 region is operatively coupled to a first member of an interactive pair of labels; and
a second oligonucleotide which is complementary to said first oligonucleotide and is operatively coupled to a second member of an interactive pair of labels, wherein said first and said second members of said pair of interactive labels interact when said first oligonucleotide and said second oligonucleotide hybridize, and do not interact when said first oligonucleotide and said second oligonucleotide dissociate.
12. The oligonucleotide pair of claim 11, wherein said second oligonucleotide is complementary to the 5\u2032 region and the 3\u2032 region of said first oligonucleotide.
13. The oligonucleotide pair of claim 11, wherein said second oligonucleotide is complementary to the 5\u2032 region of said first oligonucleotide, but is non-complementary to the 3\u2032 region of said first oligonucleotide.
14. A labeled oligonucleotide pair, comprising:
a first oligonucleotide comprising a 5\u2032 region and a 3\u2032 region, wherein the 5\u2032 region is at least partially complementary to a target nucleic acid and the 3\u2032 region is non-complementary to the target nucleic acid;
a second oligonucleotide which is at least partially complementary to said first oligonucleotide; and
an interactive pair of labels, wherein a first member of said interactive pair of labels is operatively coupled to said first oligonucleotide and a second member of said interactive pair of labels is operatively coupled to said second oligonucleotide, wherein when said first oligonucleotide and said second oligonucleotide hybridize said labels interact, and when said first and second oligonucleotides dissociate said labels do not interact.
15. The labeled oligonucleotide pair of claim 14, wherein said second oligonucleotide is complementary to the 5\u2032 region and the 3\u2032 region of said first oligonucleotide.
16. The labeled oligonucleotide pair of claim 14, wherein said second oligonucleotide is complementary to the 5\u2032 region of said first oligonucleotide, but is non-complementary to the 3\u2032 region of said first oligonucleotide.
17. The labeled oligonucleotide pair of claim 14, wherein said first member of said interactive pair of labels is operatively coupled to the 3\u2032 region of said first oligonucleotide.
18. A composition for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, said composition comprising the labeled oligonucleotide pair of claim 11 or 14, and a 3\u2032-5\u2032 exonuclease.
19. The composition of claim 18, further comprising an oligonucleotide primer.
20. The composition of claim 18, further comprising a nucleic acid polymerase.
21. A labeled oligonucleotide pair, comprising:
a first oligonucleotide comprising a 5\u2032 region and a 3\u2032 region, wherein said 5\u2032 region is complementary to a target nucleic acid and the 3\u2032 region is non-complementary to the target nucleic acid; and
a second oligonucleotide comprising a 5\u2032 region and a 3\u2032 region, wherein said 3\u2032 region is complementary to said 5\u2032 region of said first oligonucleotide and a nucleic acid strand complementary to the target, and wherein said 5\u2032 region is non-complementary to the nucleic acid strand complementary to the target;
an interactive pair of labels operatively coupled to said 3\u2032 region of said second oligonucleotide, wherein said interactive pair of labels being separated by a site susceptible to FEN nuclease cleavage, thereby allowing the nuclease activity of the FEN nuclease to separate a first interactive label from a second interactive label by cleaving at said site susceptible to the FEN nuclease, thereby generating a detectable signal.
22. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising an upstream primer, a downstream probe having a 3\u2032 flap, a 3\u2032-5\u2032 exonuclease and a suitable buffer.
23. The kit of claim 22, wherein said 3\u2032-5\u2032 exonuclease is a polymerase selected from the group consisting of: Pyrococcus furiosus (Pfu) DNA polymerase, Thermococcus litoralis DNA polymerase, Themrococcus barossii DNA polymerase, Thermococcus gorgonarius DNA polymerase and E. coli DNA polymerase I.
24. The kit of claim 22, wherein said 3\u2032-5\u2032 exonuclease is thermostable.
25. The kit of claim 22, wherein a 5\u2032 region of the downstream probe is complementary to the target.
26. The kit of claim 22, wherein the downstream probe comprises at least one labeled moiety capable of providing a signal.
27. The kit of claim 22, wherein the downstream probe comprises an interactive pair of labels.
28. The kit of claim 27, wherein said interactive pair of labels comprises a quencher moiety and a fluorescent moiety.
29. The kit of claim 27, wherein at least one member of the interactive pair of labels is operatively coupled to the 3\u2032 flap of the downstream probe.
30. The kit of claim 27, wherein a first member of the interactive pair of labels is operatively coupled to the 3\u2032 flap and a second member of the interactive pair of labels is operatively coupled to the 5\u2032 region of the downstream probe.
31. A kit comprising the labeled oligonucleotide pair of claim 11 or 14, and packing materials therefore.
32. The kit of claim 31, further comprising a 3\u2032-5\u2032 exonuclease.
33. The kit of claim 31, further comprising an oligonucleotide primer.
34. The kit of claim 32, further comprising a nucleic acid polymerase.
35. A method for detecting a target nucleic acid in a sample, the method comprising:
a. contacting a sample comprising the target nucleic acid with:
a first oligonucleotide comprising a 5\u2032 region and a 3\u2032 region, wherein the 5\u2032 region is complementary to the target nucleic acid and the 3\u2032 region is non-complementary to the target nucleic acid, and wherein the 3\u2032 region is operatively coupled to a first member of an interactive pair of labels,
a second oligonucleotide which is complementary to said first oligonucleotide and is operatively coupled to a second member of an interactive pair of labels, wherein said first and said second members of said interactive pair of labels interact when said first oligonucleotide and said second oligonucleotide hybridize, and do not interact when said first oligonucleotide and said second oligonucleotide dissociate,
a 3\u2032-5\u2032 exonuclease; and
b. detecting andor measuring a signal produced from one of said members of said interactive pair of labels.
36. A method for detecting a target nucleic acid in a sample, the method comprising:
a. forming a reaction mixture by contacting a sample comprising the target nucleic acid with:
a first oligonucleotide comprising a 5\u2032 region and a 3\u2032 region, wherein the 5\u2032 region is complementary to the target nucleic acid and the 3\u2032 region is non-complementary to the target nucleic acid, and wherein the 3\u2032 region is operatively coupled to a first member of an interactive pair of labels,
a second oligonucleotide which is complementary to said first oligonucleotide and is operatively coupled to a second member of an interactive pair of labels, wherein said first and said second members of said interactive pair of labels interact when said first oligonucleotide and said second oligonucleotide hybridize, and do not interact when said first oligonucleotide and said second oligonucleotide dissociate,
a 3\u2032-5\u2032 exonuclease, and
a polymerase;
b. subjecting said reaction mixture to conditions which permit:
annealing of said first oligonucleotide to said target nucleic acid, wherein the 3\u2032 region of said first oligonucleotide forms a flap;
cleaving said flap from said first oligonucleotide with said 3\u2032-5\u2032 exonuclease, and
extending said cleaved first oligonucleotide with said polymerase, thereby generating a nucleic acid strand complementary to said target nucleic acid; and
c. detecting andor measuring a signal produced from one of said members of said interactive pair of labels.
37. A method for detecting a target nucleic acid in a sample, the method comprising:
a. forming a reaction mixture by contacting a sample comprising the target nucleic acid with:
a first oligonucleotide and a second oligonucleotide which hybridize and wherein each oligonucleotide has one member of an interactive pair of labels which interact when said first and second oligonucleotides hybridize but do not interact when said first and said second oligonucleotides dissociate, a 3\u2032-5\u2032 exonuclease;
b. subjecting said reaction mixture to conditions which permit:
annealing of said first oligonucleotide to said target nucleic acid, wherein said first oligonucleotide forms a 3\u2032 flap when annealed to said target nucleic acid, and
cleaving said 3\u2032 flap of said first oligonucleotide with said 3\u2032-5\u2032 exonuclease; and
c. detecting andor measuring a signal produced from one of said members of said interactive pair of labels.
38. A method for detecting a target nucleic acid in a sample, the method comprising:
a. forming a reaction mixture by contacting a sample comprising the target nucleic acid with:
a first oligonucleotide and a second oligonucleotide which hybridize and wherein each oligonucleotide has one member of an interactive pair of labels which interact when said first and second oligonucleotide hybridize, and do not interact when said first and said second oligonucleotides dissociate,
a 3\u2032-5\u2032 exonuclease, and
a polymerase;
b. subjecting said reaction mixture to reaction conditions which permit:
annealing of said first oligonucleotide to said target nucleic acid, wherein said first oligonucleotide forms a 3\u2032 flap when annealed to said target nucleic acid,
cleaving of said 3\u2032 flap of said first oligonucleotide by said 3\u2032-5\u2032 exonuclease,
extending said cleaved first oligonucleotide by said polymerase thereby generating a nucleic acid strand complementary to said target; and
c. detecting andor measuring a signal produced from one of said members of said interactive label.
39. The method of claim 36 or 38, wherein said nuclease and said polymerase are the same polypeptide.
40. The method of claim 36 or 38, wherein said nuclease and the polymerase are different polypeptides.
41. The method of any one of claims 35-38, wherein said nuclease is Pyrococcus furiosus (Pfu) DNA polymerase, Thermococcus litoralis DNA polymerase, Thermococcus barossii DNA polymerase, Thermococcus gorgonarius DNA polymerase and E. coli DNA polymerase I.
42. The method of any one of claims 35-38, wherein the 3\u2032 region is one nucleotide.
43. The method of any one of claims 35-38, wherein the 3\u2032 region is two nucleotides
44. The method of any one of claims 35-38, wherein the 3\u2032 region is three nucleotides.
45. The method of any one of claims 35-38, wherein the polymerase is selected from the group consisting of: Pyrococcus furiosus (Pfu) DNA polymerase, Thermococcus litoralis DNA polymerase, Themrococcus barossii DNA polymerase, Thermococcus gorgonarius DNA polymerase and E. coli DNA polymerase I.
46. The method of claim 35 or 37 wherein the 3\u2032-5\u2032 exonuclease is thermostable.
47. The method of claim 36 or 38, wherein the 3\u2032-5\u2032 exonuclease and polymerase are thermostable.
48. The method of any one of claims 35-38, wherein said 3\u2032-5\u2032 exonuclease is Pyrococcus furiosus (Pfu) polymerase.
49. The method of any one of claims 35-38, wherein the target nucleic acid is detected by detecting a change in fluorescence intensity.
50. The method of any one of claims 35-38, wherein the interactive pair of labels comprises a quencher and a fluorophore.
51. The method of any one of claims 35-38, wherein said second moiety is a fluorophore.
52. A method for detecting a target nucleic acid in a sample, the method comprising:
a. forming a reaction mixture by contacting a sample comprising the target nucleic acid with:
a first oligonucleotide comprising a 5\u2032 region and a 3\u2032 region, wherein said 5\u2032 region is complementary to the target nucleic acid and said 3\u2032 region is non-complementary to the target nucleic acid,
a second oligonucleotide comprising a 5\u2032 region and a 3\u2032 region, wherein said 3\u2032 region is complementary to said 5\u2032 region of said first oligonucleotide and a nucleic acid strand complementary to the target, and wherein said 5\u2032 region is non-complementary to the nucleic acid strand complementary to the target,
an interactive pair of labels operatively coupled to said 3\u2032 region of said second oligonucleotide, wherein said interactive pair of labels being separated by a site susceptible to FEN nuclease cleavage, thereby allowing the nuclease activity of the FEN nuclease to separate a first interactive label from a second interactive label by cleaving at said site susceptible to the FEN nuclease, thereby generating a detectable signal,
a reverse primer,
a 3\u2032-5\u2032 exonuclease,
a FEN nuclease, and
a polymerase
b. subjecting said reaction mixture to reaction conditions which permit:
annealing of said first oligonucleotide to the target, wherein said first oligonucleotide forms a 3\u2032 flap,
cleaving said 3\u2032 flap with said 3\u2032-5\u2032 exonuclease,
extending said cleaved first oligonucleotide by said polymerase thereby generating the nucleic acid strand complementary to the target,
annealing said second oligonucleotide and said reverse primer to the nucleic acid strand complementary to the target, wherein said second oligonucleotide forms a 5\u2032 flap,
extending the reverse primer, and
cleaving the 5\u2032 flap by said FEN nuclease thereby separating said interactive pair of labels generating a detectable signal; and
c. detecting andor measuring the signal generated from one of the members of said interactive labels.