Patent Application

App. No. 11/507,735

Abstract

Claims (80)

1 . A method comprising:

amplifying a nucleic acid in at least one conduit to form an amplicon, the at least one conduit comprising an inner wall;

attaching the amplicon to the inner wall to form an attached amplicon; and

detecting the attached amplicon or an attached derivative thereof, in the at least one conduit.

2 . A method comprising:

sequentially contacting an aqueous sample fluid in a conduit with a non-aqueous spacing fluid that is immiscible with the aqueous sample, to form a plurality of discrete volumes of the aqueous sample fluid separated from one another by the non-aqueous spacing fluid, the aqueous sample fluid comprising a plurality of target nucleic acid sequences, wherein at least one of the discrete volumes contains at least one target nucleic acid sequence;

amplifying the at least one target nucleic acid in the conduit to form an amplicon; and

subjecting the amplicon to a nucleic acid sequencing reaction in the conduit.

3 . The method of claim 2 , wherein subjecting the nucleic acid sequence to a sequencing reaction forms a detectable product, and the method further comprises detecting the detectable product.

4 . The method of claim 3 , wherein the detectable product is detected inside the conduit.

5 . The method of claim 3 , wherein the detectable product is detected with a flow cell.

6 . The method of claim 2 , wherein the sequencing reaction comprises a Sanger sequencing reaction.

7 . The method of claim 2 , further comprising dividing the at least on nucleic acid containing discrete volume into two or more portions before the subjecting, and wherein the sequencing reaction comprises a forward sequencing reaction involving at least one primer, in one of the portions, and a reverse sequencing reaction involving at least one primer, in another of the portions.

8 . The method of claim 2 , further comprising introducing polymerase chain reaction inactivating reagents into the conduit after the amplifying and before the subjecting.

9 . The method of claim 2 , wherein prior to amplifying less than 50% of the plurality of discrete volumes comprise a single target nucleic acid sequence.

10 . The method of claim 2 , wherein the contacting comprises continuously flowing at least one of the aqueous sample fluid and the non-aqueous spacing fluid into the conduit.

11 . The method of claim 2 , wherein the contacting comprises sequentially introducing the aqueous sample fluid and the non-aqueous spacing fluid into the conduit.

12 . A method comprising:

flowing a first fluid and a second fluid into a conduit, the first fluid and the second fluid being immiscible with respect to one another, the first fluid containing a plurality of target molecules, wherein the maximum cross-sectional dimension of the conduit is such that a plurality of discrete volumes of the first fluid are formed in the conduit and separated from one another by the second fluid and at least one of the discrete volumes of the first fluid contains a single target molecule.

13 . The method of claim 12 , wherein at least one of the discrete volumes of the first fluid is free of the target molecules.

14 . The method of claim 12 , further comprising amplifying the single target molecule in the at least one discrete volume to form an amplification product.

15 . The method of claim 14 , wherein the amplification product comprises a nucleic acid and the method further comprises subjecting the amplification product to a nucleic acid sequencing reaction.

16 . The method of claim 15 , wherein the nucleic acid sequencing reaction comprises a Sanger sequencing reaction.

17 . The method of claim 15 , further comprising dividing the at least one discrete volume into two or more portions before the subjecting, and wherein the nucleic acid sequencing reaction comprises a forward sequencing reaction involving at least one primer, in one of the portions, and a reverse sequencing reaction involving at least one primer, in another of the portions.

18 . The method of claim 12 , wherein the conduit has an inner diameter of about 300 microns or less.

19 . The method of claim 12 , wherein the first fluid comprises an aqueous fluid, and the plurality of target molecules comprises a plurality of target nucleic acids.

20 . The method of claim 12 , further comprising adding to at least one of the plurality of discrete volumes of the first fluid one or more additional aqueous reagents to form at least one extended discrete volume.

21 . A system comprising:

at least one conduit comprising an inner surface and having a maximum cross-sectional dimension;

an aqueous sample fluid introduction unit in fluid communication with the at least one conduit;

a spacing fluid introduction unit in fluid communication with the at least one conduit, the aqueous sample fluid introduction unit and the spacing fluid introduction unit comprising separate units each in fluid communication with the at least one conduit; and

a control unit adapted to flow an aqueous sample fluid and a spacing fluid from the aqueous sample fluid introduction unit and the spacing fluid introduction unit, respectively, and adapted to inject volumes of aqueous sample fluid and spacing fluid that respectively form discrete volumes in the at least one conduit wherein each discrete volume comprises a dimension that is at least about 95% of the maximum cross-sectional dimension.

22 . The system of claim 21 , wherein the inner surface comprises an attached linker moiety.

23 . The system of claim 21 , further comprising an aqueous sample fluid source in fluid communication with the aqueous sample fluid introduction unit, and a spacing fluid source in fluid communication with the spacing fluid introduction unit.

24 . The system of claim 21 , further comprising a detection device adapted to detect a component of the aqueous sample fluid source, or a product thereof, while in the conduit.

25 . The system of claim 24 , wherein the detection device comprises at least one of a spectrophotometer, a refractive index detector, and a fluorometer.

26 . The system of claim 21 , further comprising a thermal-cycling device adapted to thermally cycle an aqueous sample fluid in the at least one conduit.

27 . The system of claim 21 , wherein the conduit comprises a conduit having an inner diameter and the inner diameter is about 300 microns or less.

28 . The system of claim 27 , wherein the inner diameter is about 50 microns or less.

29 . A system comprising:

a plurality of sample discrete volumes in a conduit, the discrete volumes being spaced-apart from one another by a spacing fluid that is immiscible with respect to the discrete volumes; and

at least one fiducial marker, the at least one fiducial marker identifying at least one volume of the discrete volumes.

30 . A system according to claim 29 , wherein the at least one fiducial marker comprises a first spacing between two adjacent sample discrete volumes of the plurality of sample discrete volumes that differs from a spacing between two or more other adjacent sample discrete volumes of the plurality.

31 . A system according to claim 29 , wherein the at least one fiducial marker comprises an aqueous discrete volume that is immiscible with the spacing fluid and contains one or more reporter molecules that are not present in the sample discrete volumes.

32 . A system according to claim 29 , wherein the at least one fiducial marker comprises two or more aqueous discrete volumes that are each immiscible with the spacing fluid and that each contain one or more reporter molecules, wherein the concentrations of the reporter molecules are different in the two or more aqueous discrete volumes.

33 . A system according to claim 29 , wherein the at least one fiducial marker comprises two or more aqueous discrete volumes that are each immiscible with the spacing fluid and that each contain one or more reporter molecules, wherein each of the two or more aqueous discrete volumes contains at least one reporter molecule that is different from at least one reporter molecule in at least one other of the two or more aqueous discrete volumes.

34 . A system comprising a first set of discrete volumes and a second set of discrete volumes, in a conduit, the discrete volumes of each set being spaced-apart from one another by a spacing fluid that is immiscible with respect to the discrete volumes; wherein the discrete volumes of the first set each have a first length, the discrete volumes of the second set each have a second length, and the first length and second length are different from one another.

35 . The system of claim 34 , further comprising at least one additional set of discrete volumes spaced-apart from one another by the spacing fluid wherein each additional set of discrete volumes is immiscible with the spacing fluid and each discrete volume of each respective additional set has a length that differs from the first length and the second length.

36 . A method comprising:

generating, in a conduit, a plurality of discrete volumes, spaced apart from one another by a spacing fluid that is immiscible with the discrete volumes, at least one of the discrete volumes containing a single target nucleic acid sequence, the target nucleic acid sequence having a first universal sequence of a universal sequence pair ligated to one end thereof and the second universal sequence of a universal sequence pair ligated to the other end thereof; and

reacting the target nucleic acid sequence with the universal primers corresponding to the universal sequence pair, in the respective discrete volume.

37 . The method of claim 36 , further comprising:

thermal-cycling the plurality of discrete volumes.

38 . The method of claim 36 , further comprising:

adding nucleic acid sequencing reagents to the discrete volumes.

39 . The method of claim 36 , further comprising:

adding a double stranded nucleic acid-detecting reactant to the discrete volumes.

40 . A method comprising:

generating, in a conduit, a plurality of discrete volumes, spaced apart from one another by a spacing fluid that is immiscible with the discrete volumes, at least one of the discrete volumes containing a plurality of target nucleic acid sequences and a plurality of pairs of tailed sequence-specific primers.

41 . The method of claim 40 , wherein

one or more of the pairs of tailed sequence-specific primers comprise identification sequence tags ligated thereto.

42 . The method of claim 40 , further comprising:

combining at least one of the discrete volumes with a respective pair of two different universal primers, wherein each respective pair of universal primers is complementary to a pair of the tailed sequence-specific primers of the plurality of pairs of tailed sequence-specific primers, and each respective pair of universal primers is unique and differs from the other respective pairs of universal primers.

43 . A nucleic acid identifying system comprising:

an identification sequence-tagged tailed forward primer comprising a first identification sequence tag, a forward universal primer sequence, and a sequence-specific forward primer; and

a tailed reverse primer comprising, a reverse universal primer sequence, and a sequence specific reverse primer;

wherein the sequence-specific forward primer and the sequence specific reverse primer are adapted to participate in a nucleic acid amplification reaction of a specific nucleic acid sequence.

44 . The nucleic acid identifying system of claim 43 , wherein

the tailed reverse primer further comprises a second identification sequence tag.

45 . The nucleic acid identifying system of claim 43 , wherein

the identification sequence-tagged tailed forward primer further comprises a 3′ end comprising a spacer nucleotide sequence.

46 . The nucleic acid identifying system of claim 43 , further comprising nucleic acid amplification reagents.

47 . The nucleic acid identifying system of claim 43 , wherein the forward universal sequence and the reverse universal sequence are artificially designed sequences.

48 . A method of nucleic acid sample tracking, comprising:

providing a tailed forward primer comprising a 5′ end, an identification sequence comprising a 3′ end, and a splint;

annealing the splint to the tailed forward primer and the identification sequence such that the end of the tailed forward primer and the end of the identification nucleic acid sequence are disposed adjacent to each other; and

ligating the tailed forward primer to the identification sequence, thereby forming an identification sequence tailed forward primer.

49 . The method of claim 48 , further comprising:

performing a nucleic acid amplification using the identification sequence tailed forward primer.

50 . The method of claim 48 , wherein the identification sequence tailed forward primer further comprises an identification tag sequence.

Assignments (11)