1. A method for detecting a RNA molecule in a sample, comprising:
combining the sample with at least one first adaptor, at least one second adaptor, and a polypeptide comprising double-strand specific RNA ligase activity to form a ligation reaction composition in which the at least one first adaptor and the at least one second adaptor are ligated to the RNA molecule of the sample to form a ligated product in the same ligation reaction composition,
wherein the at least one first adaptor comprises:
a first oligonucleotide having a length of 10 to 60 nucleotides and comprising at least two ribonucleosides on the 3\u2032-end, and
a second oligonucleotide comprising a nucleotide sequence substantially complementary to the first oligonucleotide and further comprising a single-stranded 5\u2032 portion of 1 to 8 nucleotides when the first oligonucleotide and the second oligonucleotide are duplexed,
wherein the at least one second adaptor comprises:
a third oligonucleotide having a length of 10 to 60 nucleotides and comprising a 5\u2032 phosphate group, and
a fourth oligonucleotide comprising a nucleotide sequence substantially complementary to the third oligonucleotide and further comprising a single-stranded 3\u2032 portion of 1 to 8 nucleotides when the third oligonucleotide and the fourth oligonucleotide are duplexed,
wherein the single-stranded portions independently have a degenerate nucleotide sequence, or a sequence that is complementary to a portion of the RNA molecule,
wherein the first and third oligonucleotides have a different nucleotide sequence;
wherein the RNA molecule hybridizes with the single-stranded portion of the at least one first adaptor and the single-stranded portion of the at least one second adaptor; and
detecting the RNA molecule of the ligated product or a surrogate thereof.
2. The method of claim 1 wherein detecting the RNA molecule or a surrogate thereof comprises:
combining the ligated product with
i) a RNA-directed DNA polymerase,
ii) a DNA polymerase comprising DNA dependent DNA polymerase activity and RNA dependent DNA polymerase activity, or
iii) a RNA-directed DNA polymerase and a DNA-directed DNA polymerase,
reverse transcribing the ligated product to form a reverse transcribed product,
digesting at least some of the ribonucleosides from the reverse transcribed product with ribonuclease H to form an amplification template,
combining the amplification template with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase when the ligated product is combined as in i), to form an amplification reaction composition,
cycling the amplification reaction composition to form at least one amplified product, and
determining the sequence of at least part of the amplified product, thereby detecting the RNA molecule.
3-11. (canceled)
12. The method of claim 1, wherein the sample comprises a plurality of RNA fragments and the detecting comprises determining an expression profile.
13. The method of claim 12, wherein a concentration of at least one species of RNA molecule is depleted prior to forming the ligated product.
14-16. (canceled)
17. A method for detecting an RNA molecule, comprising:
combining the RNA molecule with at least one first adaptor, at least one second adaptor, and a double-strand specific ligase to form a ligation reaction composition, wherein the at least one first adaptor comprises a first oligonucleotide comprising at least two ribonucleosides on the 3\u2032-end and a second oligonucleotide that comprises a single-stranded portion when the first oligonucleotide and the second oligonucleotide are hybridized together, and wherein the at least one second adaptor comprises a third oligonucleotide that comprises a 5\u2032 phosphate group and a fourth oligonucleotide that comprises a single-stranded portion when the third oligonucleotide and the fourth oligonucleotide are hybridized together,
ligating the at least one first adaptor and the at least one second adaptor to the RNA molecule to form a ligated product, wherein the first adaptor and the second adaptor are ligated to the RNA molecule in the same ligation reaction composition,
combining the ligated product with an RNA-directed DNA polymerase,
reverse transcribing the ligated product to form a reverse transcribed product,
digesting at least some of the ribonucleosides from the reverse transcribed product with a ribonuclease H (RNase H) to form an amplification template,
combining the amplification template with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase to form an amplification reaction composition,
cycling the amplification reaction composition to form an amplified product, and determining the sequence of at least part of the amplified product, thereby detecting the RNA molecule.
18. The method of claim 17, wherein the single-stranded portion of the first adaptor, the single-stranded portion of the second adaptor, or the single-stranded portion of the first adaptor and the single-stranded portion of the second adaptor, comprise degenerate sequences.
19. The method of claim 17, wherein the at least two ribonucleosides of the first oligonucleotide comprises at least fifteen ribonucleosides.
20. The method of claim 17, wherein the second adaptor comprises at least one reporter group.
21. The method of claim 17, wherein at least one of the amplified products comprises an identification sequence.
22. The method of claim 21, wherein at least one of the at least one forward primer, at least one of the at least one reverse primer, or both, comprise an identification sequence.
23. The method of claim 21, wherein at least one first adaptor, at least one second adaptor, or at least one first adaptor and at least one second adaptor comprises an identification sequence.
24-27. (canceled)
28. The method of claim 17, wherein the double strand-specific ligase comprises an Rnl2 family ligase.
29. The method of claim 28, wherein double strand-specific ligase comprises bacteriophage T4 RNA ligase 2 (Rnl2).
30. The method of claim 17, wherein the single-stranded portion of the first adaptor, the single-stranded portion of the second adaptor, or the single-stranded portion of the first adaptor and the single-stranded portion of the second adaptor comprise a sequence-specific region to selectively hybridize with a corresponding RNA molecule.
31-33. (canceled)
34. A kit comprising: a plurality of first adaptor species, wherein each first adaptor species comprises a different degenerate sequence, a plurality of second adaptor species, wherein each second adaptor species comprises a different degenerate sequence, a ligase of the Rnl2 family, a RNA-directed DNA polymerase, a plurality of different first primer species, a DNA-directed DNA polymerase and RNase H (EC 3.1.26.4).
35. The kit of claim 34, further comprising tobacco acid pyrophosphatase.
36. A kit comprising: a plurality of first adaptor species, wherein at least some of the first adaptor species comprise a degenerate sequence, a plurality of second adaptor species, wherein at least some of the second adaptor species comprise a degenerate sequence, a polypeptide comprising double-strand specific RNA ligase activity, a DNA polymerase, at least one primer species, and ribonuclease H.
37. The kit of claim 36, wherein the DNA polymerase comprises an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase.
38. The kit of claim 37, further comprising tobacco acid pyrophosphatase.
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 beverage maker for selectively making hot or chilled beverages, the beverage maker comprising:
a housing having a base section and a body section,
a liquid reservoir contained within the housing;
a heating element contained within the housing,
wherein the heating unit is operably coupled to the liquid reservoir; and
a cooling unit contained operably coupled to a brewing unit contained within the housing, the cooling unit comprising at least a motor, a compressor, and a length of conduit,
wherein the brewing unit has a cold brewing unit containing at least one beverage pathway therethrough.
2. The beverage maker of claim 1 further comprising an insulative material at least partially surrounding the cold brewing unit.
3. The beverage maker of claim 1 further comprising a resting plate in the base section of the housing with the resting plate being capable of being selectively warmed or cooled.
4. The beverage maker of claim 1 wherein the cold brewing unit is removable.
5. The beverage maker of claim 1 further comprising a plurality of touch sensitive buttons.
6. The beverage maker of claim 5 wherein at least one of the plurality of touch sensitive buttons controls the temperature of a brewed beverage.
7. A beverage maker for selectively making hot or chilled beverages, the beverage maker comprising:
a housing having a base section and a body section with the base section;
a cooling unit,
wherein the cooling unit has a motor, a compressor, and a length of conduit coupled thereto, the length of conduit having a coolant contained therein;
a liquid reservoir sized to hold an amount of a liquid;
a heating element coupled to the liquid reservoir;
a brewing unit operably connected to the cooling unit,
wherein the brewing unit has a cold brewing unit, the cold brewing unit having a top, bottom, and at least one sidewall therebetween,
wherein the cold brewing unit is encapsulated in an insulative material,
wherein at least one aperture is located in the top of the cold brewing unit and at least one aperture is located in the bottom of the cold brewing unit,
wherein the top and bottom aperture are coupled by at least one helical pathway contained within the cold brewing unit; and
wherein the cold brewing unit and the heating element are capable of being selectively operated thereby causing a warm or cooler beverage to be brewed by the beverage maker.
8. The beverage maker of claim 7 further comprising a power source.
9. The beverage maker of claim 7 further comprising a resting plate, wherein the resting plate is capable of being selectively warmed or cooled.
10. A method of selectively making a hot or cold beverage using a beverage maker, the method comprising the steps of:
selecting at least one brewing parameter,
wherein the at least one brewing parameter is a temperature of the hot or cold beverage;
initiating a brewing cycle,
wherein a hot brewing cycle uses hot, unchilled liquid to create a hot brewed beverage, and
wherein a cold brewing cycle chills a brewing unit thereby chilling the beverage as it passes therethrough,
wherein the brewing unit has a cold brewing unit, the cold brewing unit having a top, bottom, and at least one sidewall therebetween,
wherein there is at least one helical pathway contained therein,
wherein at least one aperture is located in the top of the cold brewing unit and at least one aperture is located in the bottom of the cold brewing unit.
11. The method of claim 10 wherein there are multiple selectable temperatures for the hot and cold beverages.
12. The method of claim 10 wherein the cold brewing unit is inactive when brewing the hot beverage.
13. The beverage maker of claim 7 wherein the length of conduit is wrapped around the cold brewing unit and covered by the insulative material.
14. The beverage maker of claim 7 wherein the length of conduit is embedded in the cold brewing unit.
15. The beverage maker of claim 7 wherein the cold brewing unit is cylindrical in shape with an aperture passing therethrough.
16. The beverage maker of claim 15 further comprising heat sinks emanating from a surface of the aperture.
17. The beverage maker of claim 7 wherein the at least one helical pathway is a double helical pathway.
18. The beverage maker of claim 7 wherein the bottom aperture of the cold brewing unit is capable of being selectively sealed thereby causing any beverage within the at least one helical pathway to be retained for an amount of time.
19. The beverage maker of claim 9 further comprising a removable ring disposed on the resting plate whereby removal of the removable ring allows for direct contact between the coolant transport and a beverage receptacle.
20. The beverage maker of claim 7 wherein a core of the cold brewing unit is a thermally conductive material.