All The Claims

1. A semiconductor device comprising:
a first silicon germanium layer formed on a single crystalline silicon substrate, the first silicon germanium layer having a concentration gradient of germanium;
a second silicon germanium layer formed on the first silicon germanium layer, the second silicon germanium layer having a concentration of germanium in a range of about 1 percent by weight to about 15 percent by weight based on the total weight of the second silicon germanium layer;
a strained silicon layer formed on the second silicon germanium layer;
an isolation layer formed at a first portion of the strained silicon layer to define an isolation region;
a gate structure formed on the isolation region of the strained silicon layer; and
sourcedrain regions formed at second portions of the strained silicon layer adjacent to the gate structure.
2. The semiconductor device of claim 1, wherein a germanium concentration in the first silicon germanium layer gradually increases from a lower portion of the first silicon germanium layer to an upper portion of the first silicon germanium layer.
3. The semiconductor device of claim 2, wherein the germanium concentration at a top portion of the first silicon germanium layer is substantially the same as that in the second silicon germanium layer.
4. The semiconductor device of claim 1,. wherein the strained silicon layer has a thickness of about 1,000 \u212b to about 5,000 \u212b.
5. The semiconductor device of claim 1, wherein the sourcedrain regions have junction depths substantially smaller than a thickness of the strained silicon layer.
6. The semiconductor device of claim 1, wherein the isolation layer has a thickness less than about 90% of a thickness of the strained silicon layer.
7. A method of manufacturing a semiconductor device comprising steps of:
forming a first silicon germanium layer on a single crystalline silicon substrate, wherein the first silicon germanium layer has a concentration gradient of germanium;
forming a second silicon germanium layer on the first silicon germanium layer, wherein the second silicon germanium layer has a concentration of germanium in a range of about 1 percent by weight to about 15 percent by weight based on the total weight of the second silicon germanium layer;
forming a strained silicon layer on the second silicon germanium layer;
forming an isolation layer at a first portion of the strained silicon layer to define an isolation region;
forming a gate structure on the isolation region of the strained silicon layer; and
forming sourcedrain regions at second portions of the strained silicon layer adjacent to the gate structure.
8. The method of claim 7, wherein the first silicon germanium layer is formed by an epitaxial growth process to provide a concentration gradient of germanium that gradually increases from a lower portion of the first silicon germanium layer to an upper portion of the first silicon germanium layer.
9. The method of claim 7, wherein the concentration of germanium in a top portion of the first silicon germanium layer is substantially the same as that in the second silicon germanium layer.
10. The method of claim 7, wherein the sourcedrain regions are formed by an ion implantation process to have junction depths substantially smaller than a thickness of the strained silicon layer.
11. The method of claim 7, wherein the strained silicon layer is formed to a thickness of about 1,000 \u212b to about 5,000 \u212b.
12. The method of claim 7, wherein the steps of forming the isolation layer further comprises steps of:
forming a buffer oxide layer pattern and a hard mask pattern on the strained silicon layer;
forming a trench at an upper portion of the strained silicon layer by partially etching the strained silicon layer using the hard mask pattern as an etching mask; and
forming the isolation layer to fill up the trench.
13. The method of claim 12, further comprising a step of cleaning the strained silicon layer using a cleaning solution after the isolation layer has been formed.
14. The method of claim 12, wherein the isolation layer is formed to have a thickness less than about 90% of a thickness of the strained silicon layer.
15. A transistor comprising:
a first silicon germanium layer formed on a single crystalline silicon substrate, the first silicon germanium layer having a concentration gradient of germanium that ranges from substantially zero at an interface between the first silicon germanium layer and the silicon substrate up to a germanium concentration that is substantially identical to that in a second silicon germanium layer at an interface between the first and the second silicon germanium layers;
a second silicon germanium layer formed on the first silicon germanium layer, the second silicon germanium layer having a concentration of germanium in a range of about 1 percent by weight to about 15 percent by weight based on the total weight of the second silicon germanium layer;
a strained silicon layer formed on the second silicon germanium layer;
an isolation layer formed at a first portion of the strained silicon layer to define an isolation region;
a gate structure formed on the isolation region of the strained silicon layer; and
sourcedrain regions formed at second portions of the strained silicon layer adjacent to the gate structure.
16. The transistor of claim 15, wherein the strained silicon layer has a thickness of about 1,000\u212b to about 5,000 \u212b.
17. The transistor of claim 15, wherein the sourcedrain regions have junction depths substantially smaller than a thickness of the strained silicon layer.
18. The transistor of claim 15, wherein the isolation layer has a thickness less than about 90% of a thickness of the strained silicon layer.

The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.

What is claimed is:

1. A motor defining an axial direction, the motor comprising:
a plurality of core pairs, each of the core pairs consisting of an inner core and an outer core, arranged next to each other along the axial direction such that the inner cores are in contact with each other;
a coil wound around each of the core pairs; and
a case formed from a magnetic material that covers the coils wherein the case is welded to at least the inner cores to form two independent magnetic circuits formed by the inner cores, the case and the outer cores.
2. A motor according to claim 1, wherein the case is welded to the outer cores.
3. A motor according to claim 1, wherein each of the inner cores and each of the outer cores has teeth-like poles;
the teeth-like poles on the inner cores and the teeth-like poles on the outer cores are alternately disposed to face a rotor magnet of a rotor that is disposed inside the plurality of core pairs; and
the case is commonly affixed to outer circumference sections of the inner cores and outer cores that form the plurality of core pairs.
4. A motor according to claim 3, wherein the case is formed from a curled thin plate.
5. A motor according to claim 4, further comprising connection terminals to supply current to the coils connected to the inner cores and the outer cores, wherein the case has an arc-shape to leave an opening for the connection terminals.
6. A motor according to claim 5, wherein the arc-shaped case has end sections, and the case and the inner cores are welded at welding spots at the end sections of the arc-shaped case and at a midpoint in the circumferential direction between the end sections of the arc-shaped case.

1. An electronic device comprising:
a memory for storing user input symbols;
a plurality of key input regions that display a subset of the user input symbols available in the memory;
motion detection circuitry configured to detect motion of the electronic device and generate an output signal corresponding to the detected motion; and
a processor coupled to the motion detection circuitry, the plurality of key input regions and the memory, wherein the processor processes the output signal of the motion detection circuitry and dynamically displays the subset of user input symbols on the plurality of key input regions based on the output signal of the motion detection circuitry.
2. The electronic device of claim 1, wherein the motion detection circuitry includes an accelerometer.
3. The electronic device of claim 1, wherein the output signal of the motion detection circuitry corresponds to a relative tilt angle of the electronic device.
4. The electronic device of any of claims 1-3, wherein the output signal of the motion detection circuitry corresponds to a rate of tilt associated with the electronic device.
5. The electronic device of claim 4, wherein the plurality of key input regions are dynamically updated at an update rate that corresponds to the rate of tilt detected.
6. The electronic device any one of claims 1-5, wherein each of the plurality of key input regions include a discrete display that is operable to display one or more user input symbols.
7. The electronic device of any one of claim 1-5, wherein the plurality of key input regions share a common display and each key input region is operable to display one or more user input symbols.
8. The electronic device of any one of claims 1-7, wherein available user input symbols for display on the plurality of key input regions are based on execution of an application by the electronic device.
9. The electronic device of any one of claims 1-8, wherein the user input symbols include symbols that represent functions such that upon selection of the user input symbol that represents a function, the corresponding function is performed by the processor.
10. The electronic device of any one of claims 1-9, wherein the plurality of key input regions are used for entering a telephone number and text input for a message.
11. The electronic device of any one of claims 1-10, wherein a single user input symbol is displayed in each of the plurality of key input regions.
12. The electronic device of any one of claims 1-11, wherein the plurality of key input regions are mapped in the memory as a template that corresponds to a conventional QWERTY keyboard.
13. The electronic device of 12, wherein the display displays the template of user input symbols for each of the plurality of key input regions.
14. A method of entering symbols in an electronic device, the method comprising:
detecting a tilt angle andor a tilt rate of an electronic device by an accelerometer housed within the electronic device, wherein the accelerometer outputs an output signal corresponding to the detected tilt andor tilt rate; and
displaying a subset of user input symbols stored in a memory of the electronic device on a plurality of key input regions based on the detected tilt andor tilt rate of the electronic device, wherein each key input region displays one user input symbol.
15. The method of claim 14 further including processing the detected tilt angle andor tilt rate to determine a mapping of the user input symbols in the memory to each of the plurality of key input regions.
16. The method of any one of claims 14-15 further including receiving a user input at one of the key input regions for selection of the user input symbol.
17. The method of any one of claim 14-16 further detecting the tilt angle andor tilt rate of the electronic device after selection of the user input symbol and dynamically updating the plurality of key input regions based upon the detected tilt angle andor tilt rate.
18. The method of claim 17, wherein the dynamic update occurs by processing the detected tilt angle andor tilt rate to determine a mapping of the user input symbols in the memory to update each of the plurality of key input regions.
19. The method of any one of claims 14-18, wherein the displayed subset of user input symbols is based at least partially on execution of an application program by the electronic device.
20. A computer program stored on a machine readable medium in an electronic device, the program being suitable for dynamically displaying user input symbols on a plurality of key input regions by processing information received from motion detection circuitry to determine a tilt angle andor a rate of tilt of the associated device, wherein the displayed user input symbols are indicative of the relative motion detected by the motion detection circuitry.

The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.

What is claimed is:

1. A method for detecting multiple reiterated oligonucleotides from a target DNA or RNA polynucleotide, said method comprising:
(a) hybridizing an initiator with a single stranded target polynucleotide
(b) incubating said target polynucleotide and initiator with an RNA-polymerase, and a terminator;
(c) synthesizing multiple oligonucleotides from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple reiterative oligonucleotides; and
(d) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts of a polynucleotide of interest.
2. The method of claim 1, further comprising detecting or quantifying said reiteratively synthesized oligonucleotide by modifying a nucleoside or nucleotide in at least one of the members selected from the group consisting of said terminator, and said initiator.
3. The method of claim 2, wherein said modifying comprises incorporating a label moiety.
4. The method of claim 3, wherein said label moiety comprises a fluorophore moiety.
5. The method of claim 4, wherein said fluorophore moiety comprises a fluorescent energy donor and a fluorescent energy acceptor wherein said moiety is detected or quantified by fluorescence resonance energy transfer.
6. The method of claim 1, wherein said polymerase is selected from the group consisting of: a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase and a modified RNA-polymerase, and a primase.
7. The method of claim 6, wherein said polymerase comprises an RNA polymerase derived from one of E. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S. typhimurium bacteriophage SP6.
8. The method of claim 1, wherein said initiator is an RNA primer.
9. The method of claim 1, wherein said initiator comprises a molecule selected from the group consisting of: nucleosides, nucleoside analogs, 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, greater than 250 nucleotides, and nucleotide analogs.
10. The method of claim 1, wherein said abortive oligonucleotides being synthesized are one of the lengths selected from the group consisting of: about 2 to about 26 nucleotides, about 26 to about 50 nucleotides, and about 50 nucleotides to about 100 nucleotides, and greater than 100 nucleotides.
11. The method of claim 1, wherein said incubating further comprises a target site probe specific for a region on said single-stranded target polynucleotide.
12. The method of claim 1, wherein said chain terminator comprises a nucleotide analog.
13. A method of detecting multiple reiterated oligonucleotides from a target DNA or RNA polynucleotide, said method comprising:
(a) hybridizing an initiator to a single-stranded target polynucleotide;
(b) incubating said target polynucleotide and initiator with a target site probe, an RNA-polymerase, and a terminator, wherein said target site probe hybridizes with said target polynucleotide;
(c) synthesizing an oligonucleotide transcript that is complementary to said target site from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple reiterative oligonucleotide transcripts; and
(d) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.
14. The method of claim 13, wherein said target site probe size is selected from the group consisting of: about 20 to about 50 nucleotides, about 51 to about 75 nucleotides, about 76 to about 100 nucleotides and greater than 100 nucleotides.
15. The method of claim 13, further comprising detecting or quantifying said reiteratively synthesized oligonucleotide by modifying a nucleotide in at least one of the members selected from the group consisting of said terminator, said initiator, and said target-site probe.
16. The method of claim 15, wherein said modifying comprises incorporating a label moiety.
17. The method of claim 16, wherein said label moiety comprises a fluorophore moiety.
18. The method of claim 17, wherein said fluorophore moiety comprises a fluorescent energy donor and a fluorescent energy acceptor wherein said moiety is detected or quantified by fluorescence resonance energy transfer.
19. The method of claim 13, wherein said polymerase is selected from the group consisting of: a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase and a modified RNA polymerase, and a primase.
20. The method of claim 19, wherein said polymerase comprises an RNA polymerase derived from one of E. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S. typhimurium bacteriophage SP6.
21. The method of claim 13, wherein said initiator is an RNA primer.
22. The method of claim 13, wherein said initiator comprises nucleotides selected from the group consisting of: 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, and greater than 250 nucleotides
23. The method of claim 13, wherein said abortive oligonucleotides being synthesized are one of the lengths selected from the group consisting of: about 2 to about 26 nucleotides, about 26 to about 50 nucleotides and about 50 nucleotides to about 100 nucleotides, and greater than 100 nucleotides.
24. The method of claim 13, wherein said mixture in a further comprises a target site probe specific for a region on said single-stranded target polynucleotide.
25. The method of claim 13, wherein said chain terminator comprises a nucleotide analog.
26. A method for detecting methylated cytosine residues at CpG sites in a target polynucleotide, comprising:
(a) deaminating a single-stranded target DNA sequence under conditions which convert unmethylated cytosine residues to uracil residues while not converting methylated cytosine residues to uracil;
(b) hybridizing an initiator with a single stranded target polynucleotide;
(c) incubating said deaminated target polynucleotide and said initiator with a terminator, and an RNA-polymerase, wherein at least one of said initiator, or terminator is modified to enable detection of the CG sites;
(d) synthesizing an oligonucleotide transcript that is complementary to said CG sites from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript thereby synthesizing multiple reiterative oligonucleotide transcripts; and
(e) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.
27. A method for detecting methylated cytosine residues at a CpG site in a target gene, said method comprising:
(a) deaminating a single-stranded target DNA polynucleotide under conditions which convert unmethylated cytosine residues to uracil residues while not converting methylated cytosine residues to uracil;
(b) hybridizing a target site probe with said single stranded target polynucleotide,
(c) incubating said target polynucleotide and target site probe with, an initiator, a terminator, and an RNA-polymerase, wherein at least one of said initiator, or said terminator are complementary to the CpG site;
(d) synthesizing an oligonucleotide transcript that is complementary to said target site from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides, thereby synthesizing multiple reiterative oligonucleotide transcripts; and
(e) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.
28. The method of claim 27, wherein said target site probe size is selected from the group consisting of: about 5-19; about 20 to about 50 nucleotides, about 51 to about 75 nucleotides, about 76 to about 100 nucleotides and greater than 100 nucleotides.
29. The method of claim 27, further comprising detecting or quantifying said reiteratively synthesized oligonucleotide by modifying a nucleotide in at least one of the members selected from the group consisting of said terminator, and said initiator.
30. The method of claim 29, wherein said modifying comprises incorporating a label moiety.
31. The method of claim 30, wherein said label moiety comprises a fluorophore moiety.
32. The method of claim 31, wherein said fluorophore moiety comprises one of a fluorescent energy donor and a fluorescent energy acceptor wherein said moiety is detected or quantified by fluorescence resonance energy transfer.
33. The method of claim 27, wherein said polymerase is selected from the group consisting of: a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase and a modified RNA polymerase, and a primase.
34. The method of claim 27, wherein said polymerase comprises an RNA polymerase derived from one of E. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S. typhimurium bacteriophage SP6.
35. The method of claim 27, wherein said initiator is an RNA primer.
36. The method of claim 27, wherein said initiator comprises nucleotides selected from the group consisting of: 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, and greater than 250 nucleotides
37. The method of claim 27, wherein said abortive oligonucleotides being synthesized are one of the lengths selected from the group consisting of: about 2 to about 26 nucleotides, about 26 to about 50 nucleotides and about 50 nucleotides to about 100 nucleotides, and greater than 100 nucleotides.
38. The method of claim 27, wherein said chain terminator comprises a nucleotide analog.
39. The method of claim 26 or 27, wherein deaminating a single-stranded target DNA sequence comprises treating said single-stranded target DNA sequence with sodium bisulfite.
40. The method of claim 27, wherein said target site probe and said target DNA sequence form a bubble complex comprising a first double-stranded region upstream of said target CpG site, a single-stranded region comprising said target CpG site, and a second double-stranded region downstream of said target CpG site.
41. A method for detecting the presence or absence of mutations in a target DNA sequence, the method comprising
(a) hybridizing a target site probe to a single-stranded DNA polynucleotide, wherein said DNA polynucleotide may contain a mutation relative to a normal or wild type gene;
(b) incubating said target polynucleotide and target-site probe with an RNA-polymerase, an initiator, and a terminator;
(c) synthesizing an oligonucleotide transcript from said target polynucleotide that is complementary to a target mutation site, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple abortive reiterative oligonucleotides; and
(d) determining the presence or absence of a mutation by detecting or quantifying said reiteratively synthesized oligonucleotides transcribed from said target DNA polynucleotide.
42. The method of claim 41, wherein said target site probe size is selected from the group consisting of: about 20 to about 50 nucleotides, about 51 to about 75 nucleotides, about 76 to about 100 nucleotides and greater than 100 nucleotides.
43. The method of claim 41, further comprising detecting or quantifying said reiteratively synthesized oligonucleotide by modifying a nucleotide in at least one of the members selected from the group consisting of said terminator, and said initiator.
44. The method of claim 43, wherein said modifying comprises incorporating a label moiety.
45. The method of claim 44, wherein said label moiety comprises a fluorophore moiety.
46. The method of claim 45, wherein said fluorophore moiety comprises a fluorescent energy donor and a fluorescent energy acceptor wherein said moiety is detected or quantified by fluorescence resonance energy transfer.
47. The method of claim 41, wherein said polymerase is selected from the group consisting of: a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase and a modified RNA polymerase, and a primase.
48. The method of claim 47, wherein said polymerase comprises an RNA polymerase derived from one of E. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S. typhimurium bacteriophage SP6.
49. The method of claim 41, wherein said abortive oligonucleotides being synthesized are one of the lengths selected from the group consisting of: about 2 to about 26 nucleotides, about 26 to about 50 nucleotides and about 50 nucleotides to about 100 nucleotides, and greater than 100 nucleotides.
50. The method of claim 41, wherein said chain terminator comprises a nucleotide analog.
51. The method of claim 41, wherein said mutation is selected from the group consisting of: a deletion, an insertion, a substitution, a chromosomal rearrangement, and a single nucleotide polymorphism.
52. The method of claim 41, wherein said initiator comprises nucleotides selected from the group consisting of: 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, and greater than 250 nucleotides
53. The method of claim 41, wherein said target site probe and said target DNA sequence form a bubble complex comprising a first double-stranded region upstream of said target mutation site, a single-stranded region comprising said target mutation site, and a second double-stranded region downstream of said target mutation site.
54. A method for detecting mutations in a target DNA polynucleotide, said method comprising:
(a) immobilizing a capture probe designed to hybridize with said target DNA polynucleotide;
(b) hybridizing said capture probe to said target DNA polynucleotide, wherein said DNA polynucleotide may contain a mutation relative to a normal or wild type gene;
(c) incubating said target polynucleotide with an RNA-polymerase, an initiator, and a terminator;
(d) synthesizing an oligonucleotide transcript that is complementary to a target site from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple abortive reiterative oligonucleotide transcripts; and
(e) determining the presence or absence of a mutation by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts from said target DNA polynucleotide.
55. A method for detecting DNA or RNA in a test sample, said method comprising:
(a) hybridizing a single stranded target polynucleotide with an abortive promoter cassette comprising a sequence that hybridizes to the single stranded target polynucleotide, and a region that can be detected by transcription by a polymerase;
(b) incubating said target polynucleotide with an RNA-polymerase, an initiator, and a terminator;
(c) synthesizing an oligonucleotide transcript that is complementary to the initiation start site of the APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides, thereby synthesizing multiple reiterative oligonucleotide transcripts; and
(d) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.
56. A method for detecting the presence of pathogens in a test sample, said method comprising the steps of:
(a) hybridizing a single stranded target pathogen polynucleotide in said test sample with an abortive promoter cassette comprising a region that can be detected by transcription by a polymerase;
(b) incubating said target polynucleotide and initiator with an RNA-polymerase, and a terminator;
(c) synthesizing an oligonucleotide transcript that is complementary to initiation start site of the APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple abortive reiterative oligonucleotide transcripts; and
(d) determining the presence of a pathogen by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts synthesized from said test sample.
57. The method of any one of claims 54-56, further comprising detecting or quantifying said reiteratively synthesized oligonucleotide transcript by modifying a nucleotide in at least one of the members selected from the group consisting of said terminator, and said initiator.
58. The method of claim 57, wherein said modifying comprises incorporating a label moiety.
59. The method of claim 58, wherein said label moiety comprises a fluorophore moiety.
60. The method of claim 59, wherein said fluorophore moiety comprises a fluorescent energy donor and a fluorescent energy acceptor wherein said moiety is detected or quantified by fluorescence resonance energy transfer.
61. The method of any one of claims 54-56, wherein said polymerase is selected from the group consisting of: a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase and a modified RNA polymerase, and a primase.
62. The method of claim 61, wherein said polymerase comprises an RNA polymerase derived from one of E. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S. typhimurium bacteriophage SP6.
63. The method of any one of claims 54-56, wherein said abortive oligonucleotides being synthesized are one of the lengths selected from the group consisting of: about 2 to about 26 nucleotides, about 26 to about 50 nucleotides and about 50 nucleotides to about 100 nucleotides.
64. The method of any one of claims 54-56, wherein said chain terminator comprises a nucleotide analog.
65. The method of claim 54 or 55, wherein said initiator comprises nucleotides selected from the group consisting of: 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, and greater than 250 nucleotides
66. The method of any one of claim 56, wherein said single-stranded target polynucleotide is one of DNA and RNA.
67. The method of any one of claims 54-56, wherein said inititator is one of RNA.
68. The method of claim 56, wherein said initiator comprises nucleotides selected from the group consisting of: 1-25 nucleotides, 25-50 nucleotides, 50-75 nucleotides, 75-100 nucleotides, 100-125 nucleotides, and 125-150 nucleotides, 150-175 nucleotides, 175-200 nucleotides, 200-225 nucleotides, and 225-250 nucleotides.
69. The method of claim 55 or claim 56, wherein said abortive promoter cassette comprises a self-complementary oligonucleotide that forms a single-stranded bubble in the presence of an RNA polymerase, wherein a region of said bubble region can be detected by transcription by said polymerase
70. The method of any one of claim 56, wherein said abortive promoter cassette comprises an APC linker which is adapted to hybridize to an end of said target pathogen polynucleotide.
71. A method for detecting pathogens in a test sample, said method comprising:
(a) immobilizing a capture probe designed to hybridize with a target DNA polynucleotide in said test sample;
(b) hybridizing said capture probe with a test sample that potentially contains said target DNA polynucleotide;
(c) hybridizing a single stranded target DNA polynucleotide in said test sample with an abortive promoter cassette comprising a region that hybridizes to the single stranded target pathogen polynucleotide, and a region that can be detected by transcription by a polymerase;
(d) incubating said target polynucleotide with an RNA-polymerase, initiator, and a terminator;
(e) synthesizing an oligonucleotide transcript that is complementary to said initiation transcription start site of APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple reiterative oligonucleotide transcripts; and
(f) determining the presence or absence of a pathogen by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.
72. A method for detecting mRNA expression in a test sample, the method comprising:
(a) hybridizing a target mRNA sequence with an abortive promoter cassette comprising a region that can be detected by transcription by a polymerase;
(b) incubating said target mRNA sequence with an RNA-polymerase, an initiator, and a terminator;
(c) synthesizing an oligonucleotide transcript that is complementary to transcription initiation start site, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple reiterative oligonucleotides; and
(d) determining the presence or absence of the mRNA by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts synthesized from said test sample.
73. The method of claim 72, further comprising:
(a) immobilizing a capture probe, wherein said probe hybridizes with a target mRNA sequence;
(b) hybridizing said capture probe with a test sample which potentially contains said target mRNA sequence; and
(c) washing a captured target mRNA sequence to remove unhybridized components of said test sample.
74. The method of claim 72, wherein modifying further comprises incorporating an independently selected label moiety into at least one of said initiator, said terminator, and said nucleotides.
75. The method of claim 74, wherein said label moiety comprises a fluorophore moiety.
76. The method of claim 75, wherein detecting comprises detecting by fluorescence resonance energy transfer and said fluorophore moiety comprises one of a fluorescent energy donor and a fluorescent energy acceptor.
77. The method of claim 72, wherein said polymerase is one of a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, and a modified polymerase, and a primase.
78. The method of claim 72, wherein said polymerase comprises an RNA polymerase derived from one of E. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S. typhimurium bacteriophage SP6.
79. The method of claim 72, wherein said initiator is one of RNA or DNA.
80. The method of claim 79, wherein said initiator comprises nucleotides selected from the group consisting of: 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, and greater than 250 nucleotides
81. The method of claim 72, wherein said abortive oligonucleotides being synthesized are one of the lengths selected from the group consisting of: about 2 to about 26 nucleotides, about 26 to about 50 nucleotides and about 50 nucleotides to about 100 nucleotides, and greater than 100 nucleotides.
82. The method of claim 72, wherein said abortive promoter cassette comprises a self-complementary oligonucleotide that forms a single-stranded bubble region comprising said target site.
83. The method of claim 72, wherein said abortive promoter cassette comprises an APC linker which is adapted to hybridize to a poly-A tail of said target mRNA sequence.
84. The method of claim 72, wherein said chain terminator comprises one of nucleotide deprivation and a nucleotide analog.
85. A method for detecting an oligonucleotide synthesized from a target DNA sequence, the method comprising:
(a) hybridizing a DNA primer with a single-stranded target DNA sequence;
(b) extending said DNA primer with a DNA polymerase and nucleotides, such that said DNA polymerase reiteratively synthesizes a nucleotide sequence; and
(c) detecting oligonucleotide comprised of repeat sequences synthesized by said DNA polymerase.
86. The method of claim 85, further comprising modifying at least one of said DNA primer and said nucleotides to enable detection of said oligonucleotide comprised of repeat sequences.
87. The method of claim 86, wherein modifying further comprises incorporating an independently selected label moiety into at least one of said DNA primer and said nucleotides.
88. The method of claim 87, wherein said label moiety comprises a fluorophore moiety.
89. The method of claim 88, wherein detecting comprises detecting by fluorescence resonance energy transfer and said fluorophore moiety comprises one of a fluorescent energy donor and a fluorescent energy acceptor.
90. The method of claim 85, wherein said DNA polymerase is selected from the group consisting of Escherichia coli DNA polymerase, T7 DNA polymerase, T4 DNA polymerase, Taq thermostable DNA polymerase, terminal transferase, and telomerase.
91. The method of claim 85, wherein said DNA primer comprises from 1 to about 25 nucleotides.
92. The method of claim 85, wherein said oligonucleotide repeat sequence comprises from about 2 to about 26 nucleotides.
93. The method of claim 85, wherein said detecting comprises hybridizing a complementary sequence to the synthesized oligonucleotide comprising repeat sequences.
94. The method of claim 93, wherein said complementary sequence is modified to comprise an independently selected label moiety.
95. The method of claim 94, wherein said label moiety comprises a fluorophore moiety
96. The method of claim 85, further comprising immobilizing said single-stranded target DNA sequence.
97. The method of claim 85, wherein immobilizing comprises hybridizing a capture probe to a portion of said single-stranded target DNA sequence.
98. The method of claim 13, wherein said target site probe and said target DNA sequence form a bubble complex comprising a first double-stranded region upstream of said target site, a single-stranded region comprising said target site, and a second double-stranded region downstream of said target site.
99. A method of producing a microarray, the method comprising:
(a) synthesizing multiple abortive oligonucleotide replicates from a target DNA sequence by the method of claim 1; and
(b) attaching said multiple abortive oligonucleotide replicates to a solid substrate to produce a microarray of said multiple abortive oligonucleotide replicates.
100. The method of any one of claims 4, 17, 31, 45, 75, 88, or 95 wherein said fluorophore moiety is selected from the group consisting of: 4-acetamido-4-isothiocyanatostilbene-2,2disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 5-(2-aminoethyl)amninonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-3-vinylsulfonyl)phenylnaphthalimide-3,5 disulfonate; N-(4-amino-1-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin, and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4,6-diaminidino-2-phenylindole (DAPI); 5,5-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid; 4,4-diisothiocyanatostilbene-2,2-disulfonic acid; 5-dimethylaminonaphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4-isothiocyanate (DABITC); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2,7-dimethoxy-45-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1pyrene; butyrate quantum dots; Reactive Red 4; rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B, sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid; terbiun chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine.
101. A method for detecting multiple reiterated oligonucleotides from a target DNA or RNA polynucleotide, said method comprising:
(a) incubating a single-stranded target polynucleotide in a mixture comprising an initiator, and an RNA-polymerase;
(b) synthesizing multiple oligonucleotide transcripts from said target polynucleotide, wherein said initiator is extended until terminated due to nucleotide deprivation, thereby synthesizing multiple abortive reiterative oligonucleotide transcripts; and
(c) detecting or quantifying said reiteratively synthesized oligonucleotides
102. A method of detecting multiple reiterated oligonucleotides from a target DNA or RNA polynucleotide, said method comprising:
(a) incubating a single-stranded target polynucleotide in a mixture comprising an initiator, an RNA-polymerase, and a target site probe, wherein said target site probe and said target polynucleotide hybridize to form a bubble complex comprising a first double-stranded region upstream of a target site, a single-stranded region comprising said target site, and a second double-stranded region downstream of said target site;
(b) synthesizing multiple oligonucleotide transcripts from said target polynucleotide, wherein said initiator is extended until terminated due to nucleotide deprivation, thereby synthesizing multiple abortive reiterative oligonucleotides; and
(c) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.
103. A method for detecting methylated cytosine residues at a CG site near a target gene, the method comprising:
(a) deaminating a single-stranded target DNA sequence under conditions which convert unmethylated cytosine residues to uracil residues while not converting methylated cytosine residues to uracil;
(b) incubating a single-stranded target polynucleotide in a mixture comprising an initiator, a terminator, an RNA-polymerase, and a target site probe;
(c) synthesizing multiple oligonucleotide transcripts from said target polynucleotide, wherein said initiator is extended until terminated due to nucleotide deprivation, thereby synthesizing multiple abortive reiterative oligonucleotide transcripts; and
(d) detecting or quantifying said reiteratively synthesized oligonucleotides
104. The method of claim 26 or 27, further comprising:
(a) immobilizing an oligonucleotide capture probe which is specific for a sequence near a CpG island related to a target gene; and
(b) hybridizing said oligonucleotide capture probe with a denatured DNA sample which potentially contains said target DNA sequence.
105. The method of claim 27, wherein said target site probe is gene specific cancer specific.
106. A method for detecting a target protein in a test sample, the method comprising:
(a) covalently attaching the target protein to an abortive promoter cassette (APC) by a reactive APC linker, wherein said APC comprises a region that can be detected by transcription by a polymerase;
(b) incubating said target protein with an RNA-polymerase, an initiator, and a terminator;
(c) synthesizing an oligonucleotide transcript that is complementary to transcription initiation start site of APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple reiterative oligonucleotide transcripts; and
(d) determining the presence or absence of the target protein by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts synthesized from said test sample.
107. The method of claim 106 further comprising immobilizing target protein by a target specific probe.
108. The method of claim 107, wherein said target specific probe is an antibody.
109. The method of claim 106, wherein said APC linker will be covalently attached to the target protein by modification of thiol-reactive or amine-reactive protein crosslinking agents.
110. The method of claim 109 wherein said protein crosslinking agents are selected from the group consisting of: maleamides, iodoacetamides, and disulfides.
111. The method of claim 106, wherein said target protein is purified or in a cell lysate.
112. A method for detecting cancer, comprising:
(a) obtaining a sample from a patient in need of detection of a cancer;
(b) deaminating the DNA under conditions which convert unmethylated cytosine residues to uracil residues while leaving the methylated cytosine residues unaltered;
(c) hybridizing an initiator to a target polynucleotide wherein said initiator is a mononucleoside, mononucleotide, binucleotide, oligonucleotide or an analog thereof;
(d) incubating said deaminated target polynucleotide and said initiator with a terminator, and an RNA-polymerase, wherein at least one of said initiator, terminator is modified to enable detection of the CG sites;
(e) synthesizing an oligonucleotide transcript that is complementary to said CG sites from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript thereby synthesizing multiple reiterative oligonucleotide transcripts;
(f) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts; and comparing the results with those obtained similarly from a control sample.
113. A method for detecting pathogens, said method comprising the steps of:
(a) obtaining a sample in need of detection of a pathogen
(b) hybridizing a single stranded target pathogen polynucleotide in said sample with an abortive promoter cassette comprising a nucleotide sequence that hybridizes to single stranded target pathogen polynucleotide, and a region that can be detected by transcription by a polymerase;
(c) incubating said target polynucleotide and initiator with an RNA-polymerase, and a terminator;
(d) synthesizing an oligonucleotide transcript that is complementary to initiation start site of the APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple abortive reiterative oligonucleotide transcripts; and
(e) determining the presence of a pathogen by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts synthesized from said sample.
114. The method of claim 113, wherein said method further comprises:
(a) immobilizing an oligonucleotide capture probe which is specific for said target pathogen polynucleotide; and
(b) hybridizing said oligonucleotide capture probe with a denatured DNA sample which potentially contains said target pathogen polynucleotide.
115. A method for synthesizing multiple reiterated oligonucleotides from a target DNA or RNA polynucleotide, said method comprising:
(a) hybridizing an initiator with a single stranded target polynucleotide
(b) incubating said target polynucleotide and initiator with an RNA-polymerase, and a terminator;
(c) synthesizing multiple oligonucleotides from said target aspolynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple reiterative oligonucleotides.
116. The method of claim 115, further comprising synthesizing oligonucleotides by modifying a nucleotide in at least one of the members selected from the group consisting of said terminator, and said initiator.
117. The method of claim 116, wherein said modifying comprises incorporating a label moiety.
118. The method of claim 117, wherein said label moiety comprises a fluorophore moiety.
119. The method of claim 118, wherein said fluorophore moiety comprises a fluorescent energy donor and a fluorescent energy acceptor.
120. The method of claim 115, wherein said polymerase is selected from the group consisting of: a DNA-dependent RNA polymerase, an RNA-dependent RNA polymerase and a modified RNA-polymerase, and a primase.
121. The method of claim 120, wherein said polymerase comprises an RNA polymerase derived from one of E. coli, E. coli bacteriophage T7, E. coli bacteriophage T3, and S. typhimurium bacteriophage SP6.
122. The method of claim 115, wherein said initiator comprises nucleotides selected from the group consisting of: 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250 nucleotides, and greater than 250 nucleotides
123. The method of claim 115, wherein said abortive oligonucleotides being synthesized are one of the lengths selected from the group consisting of: about 2 to about 26 nucleotides, about 26 to about 50 nucleotides and about 50 nucleotides to about 100 nucleotides.
124. The method of claim 115, wherein said incubating further comprises a target site probe specific for a region on said single-stranded target polynucleotide.
125. The method of claim 115, wherein said chain terminator comprises a nucleotide analog.
126. The method of any one of claims 1, 13, 26, 27, 41, 54, 55, 56, 71, 72, 101, 102, 103, 106, 112, 113, or 115, wherein said incubating further comprises in the presence of ribonucleotides.
127. The method of claim 126, wherein said ribonucleotides are modified.
128. The method of claim 127, wherein said modifying further comprises incorporating an independently selected label moiety.
129. The method of claim 128, wherein said label moiety comprises a fluorophore moiety.
130. The method of claim 112 or 113, wherein said sample is obtained from the group consisting of: animal, plant or human tissue, blood, saliva, semen, urine, sera, cerebral or spinal fluid, pleural fluid, lymph, sputum, fluid from breast lavage, mucusoal secretions, animal solids, stool, cultures of microorganisms, liquid and solid food and feedproducts, waste, cosmetics, air and water.
131. The method of any one of claims 55, 56, 71, 72, 106, or 113, wherein said abortive promoter cassette comprises two partially complementary oligonucleotides that form a bubble region.
132. The method of any one of claims 55, 56, 71, 72, 106, or 113, wherein said abortive promoter cassette comprises two complementary oligonucleotides that form a bubble region in the presence of RNA polymerase.
133. The method of any one of claims 55, 56, 71, 72, 106, or 113, wherein said abortive promoter cassette comprises one contiguous oligonucleotide to which RNA polymerase can bind to form a transcription bubble.
134. The method of claim 59 wherein said fluorophore moiety is selected from the group consisting of: 4-acetamido-4-isothiocyanatostilbene-2,2disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 5-(2-aminoethyl)amninonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-3-vinylsulfonyl)phenylnaphthalimide-3,5disulfonate; N-(4-amino-1-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin, and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4,6-diaminidino-2-phenylindole (DAPI); 5,5-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid; 4,4-diisothiocyanatostilbene-2,2-disulfonic acid; 5-dimethylaminonaphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4-isothiocyanate (DABITC); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2,7-dimethoxy-45-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl lpyrene; butyrate quantum dots; Reactive Red 4; rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B, sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid; terbiun chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine.
135. The method of any one of claims 1, 13, 26, 27, 41, 54-56, 71, 72, 85, 101-103, 106, 112, 113, or 115, wherein said initiator is selected from the group consisting of: nucleosides, nucleoside analogs, nucleotides, an nucleotide analogs.