| Número de publicación||USRE41005 E1|
|Tipo de publicación||Concesión|
| Número de solicitud||US 10/277,021|
| Fecha de publicación||24 Nov 2009|
| Fecha de presentación||17 Oct 2002|
| Fecha de prioridad||6 Nov 1996|
|También publicado como||US6133436, USRE44693|
| Número de publicación||10277021, 277021, US RE41005 E1, US RE41005E1, US-E1-RE41005, USRE41005 E1, USRE41005E1|
| Inventores||Hubert Koster, David M. Lough|
| Cesionario original||Sequenom, Inc.|
|Exportar cita||BiBTeX, EndNote, RefMan|
|Citas de patentes (136), Otras citas (101), Citada por (1), Clasificaciones (43), Eventos legales (2) |
|Enlaces externos: USPTO, Cesión de USPTO, Espacenet|
Functionalized beads for the immobilization of nucleic acids, wherein the beads are stably associated with a solid support selected from multiwell plates, arrays of pits and multiwell supports
US RE41005 E1
Novel compositions comprised of at least one bead conjugated to a solid support and further conjugated to at least one nucleic acid and preferred methods for making the novel compositions are described. As compared to “flat” surfaces, beads linked to a solid support provide an increased surface area for immobilization of nucleic acids. Furthermore, by selecting a bead with the desired functionality, a practitioner can select a functionalization chemistry for immobilizing nucleic acids, which is different from the chemistry of the solid support.
1. A composition, comprising a bead conjugated to a solid support and further conjugated to a nucleic acid, wherein the solid support is selected from the group consisting of multiwell plates, arrays of pits and multiwell supports comprising nanoliter wells.
2. A composition of claim 1, wherein the bead is made from a material selected from the group consisting of: silica gel, glass, magnet, 4—(hydroxymethyl)phenoxymethylcopoly(styrene—1% divinylbenzene) resin, chloromethylated copolystyrene—divinylbenzene resin, metal, plastic, cellulose, dextran cross-linked with epichlorohydrin, and agarose.
3. A composition of claim 1, wherein the bead is swellable.
4. A composition of claim 1, wherein the bead is nonswellable.
5. A composition of claim 1, wherein the bead is in the range of 1 to 100 μm in diameter.
6. A composition of claim 1, wherein the nucleic acid is DNA.
7. A composition of claim 1, wherein the nucleic acid is RNA.
8. A process of making a bead conjugated to a solid support and further conjugated to a nucleic acid, comprising the steps of conjugating a bead to a nucleic acid; and conjugating a bead to a solid support, wherein the solid support is selected from the group consisting of multiwell plates, arrays of pits, and multiwell supports comprising nanoliter wells.
9. A process of claim 8, wherein the bead is functionalized.
10. A process of claim 9, wherein the bead is functionalized with carboxy functional groups.
11. A process of claim 9, wherein the bead is functionalized with amino functional groups.
12. A process of claim 9, wherein the bead is conjugated to the nucleic acid prior to conjugation of the bead to the solid support.
13. A process of claim 9, wherein the bead is conjugated to the nucleic acid after the bead is conjugated to the solid support.
14. A kit, comprising:
ii) an insoluble support, and
iii) conjugation means for linking nucleic acids to the beads and the beads to the support, wherein the solid support is selected from the group consisting of arrays of pits and multiwell supports comprising nanoliter wells.
15. The kit of claim 14, wherein the solid support is selected from the group consisting of: beads, capillaries, plates, membranes, wafers, combs, pins, wafers with arrays of pits, and supports with nanoliter wells.
16. The kit of claim 14, wherein the bead is made from material selected from the group consisting of silica gel, glass, magnet, p-benzyloxybenzyl alcohol copolystyrene-divinyl benzene (DVB) resin, chlorotritylchloride copolystyrene-DVB resin, chloromethylated copolystyrene-DVB resin, metal, plastic, cellulose, cross-linked dextran, and agarose gel.
17. A composition, comprising a bead conjugated to a solid support and further conjugated to a nucleic acid, wherein conjugation is effected with a crosslinking agent and the solid support is selected from the group consisting of arrays of pits and multiwell supports comprising nanoliter wells.
18. The method of claim 8, wherein conjugation is effected with a crosslinking agent.
19. A composition, comprising a bead conjugated to a solid support and further conjugated to aThe composition of claim 1, wherein the nucleic acid molecule comprising proteincomprises a peptide nucleic acid.
20. A composition, comprising a bead conjugated to a solid support and further conjugated to a nucleic acid, wherein conjugation is effected through a photocleavable linkage, and the solid support is selected from the group consisting of arrays of pits and multiwell supports comprising nanoliter wells.
21. The composition of claim 20, wherein the linkage is cleaved by exposure to a laser.
22. The composition of claim 20, wherein the linkage is cleaved by exposure to electromagnetic radiation selected from ultraviolet, visible, infrared radiation or electromagnetic radiation generated by fluorescence or chemiluminescence, or combinations thereof.
23. A composition, comprising a bead conjugated to a solid support and further conjugated to a nucleic acid, wherein conjugation is effected through ionic linkages.
24. The composition of claim 1, wherein the solid support comprises an array of pits.
25. The composition of claim 1, wherein beads are conjugated to the support in pits on the array.
26. The composition of claim 1, wherein the solid support is a multiwell support comprising nanoliter wells.
27. The composition of claim 1, wherein beads are conjugated to the support in wells on the support.
28. The composition of claim 1, wherein conjugation of the bead to the solid support and/or conjugation of the nucleic acid to the bead is effected through an interaction comprising an ionic, covalent, polar or hydrophobic interaction.
29. The composition of claim 28, wherein interaction is an ionic interaction.
30. The composition of claim 28, wherein the interaction is a covalent interaction.
31. The composition of claim 28, wherein the interaction is a polar interaction.
32. The composition of claim 28, wherein the interaction is a hydrophobic interaction.
33. A method, comprising:
a) conjugating a bead to a solid support and further conjugating the bead to a nucleic acid, wherein the solid support is selected from the group consisting of arrays of pits and multiwell supports comprising nanoliter wells; and
b) analyzing the nucleic acid by a spectrometric method.
34. The method of claim 33, wherein the solid support comprises an array of pits.
35. The method of claim 34, wherein beads are conjugated to the support in pits on the array.
36. The method of claim 33, wherein the solid support is a multiwell support comprising nanoliter wells.
37. The method of claim 36, wherein beads are conjugated to the support in wells on the support.
38. A method, comprising:
a) providing a composition comprising a bead conjugated to a solid support and further conjugated to a nucleic acid, wherein the solid support is selected from the group consisting of arrays of pits and multiwell supports comprising nanoliter wells; and
b) analyzing the nucleic acid by a spectrometric method.
39. The method of claim 38, wherein the solid support comprises an array of pits.
40. The method of claim 39, wherein beads are conjugated to the support in pits on the array.
41. The method of claim 38, wherein the solid support is a multiwell support comprising nanoliter wells.
42. The method of claim 41, wherein beads are conjugated to the support in wells on the support.
43. The composition of claim 1, wherein conjugation of the bead to the solid support and/or conjugation of the nucleic acid to the bead is effected through an acid labile linkage.
44. The composition of claim 1, wherein the nucleic acid is single-stranded.
45. The process of claim 8, wherein the nucleic acid is single-stranded.
46. The method of claim 33, wherein the nucleic acid is single-stranded.
47. The method of claim 38, wherein the nucleic acid is single-stranded.
48. A method, comprising:
(a) contacting a target nucleic acid with beads conjugated to a solid support and further conjugated to a nucleic acid, wherein target nucleic acid that hybridizes to the nucleic acid conjugated to the beads is captured, and wherein the solid support is selected from the group consisting of arrays of pits and multiwell supports comprising nanoliter wells; and
(b) detecting captured target nucleic acid.
49. The method of claim 48, wherein the nucleic acid conjugated to the beads is single-stranded.
50. The method of claim 48, wherein the solid support comprises an array of pits.
51. The method of claim 50, wherein beads are conjugated to the support in pits on the array.
52. The method of claim 48, wherein the solid support is a multiwell support comprising nanoliter wells.
53. The method of claim 52, wherein beads are conjugated to the support in wells on the support.
54. The method of claim 48, wherein conjugation of the beads to the solid support and/or conjugation of the nucleic acid to the beads is effected through ionic, covalent, polar or hydrophobic interactions.
55. The method of claim 54, wherein conjugation of the beads to the solid support and/or conjugation of the nucleic acid to the beads is effected through ionic interactions.
56. The method of claim 54, wherein conjugation of the beads to the solid support and/or conjugation of the nucleic acid to the bead is effected through covalent interactions.
57. The method of claim 54, wherein conjugation of the beads to the solid support and/or conjugation of the nucleic acid to the bead is effected through polar interactions.
58. The method of claim 54, wherein conjugation of the beads to the solid support and/or conjugation of the nucleic acid to the beads is effected through hydrophobic interactions.
59. The method of claim 48, wherein the nucleic acid is detected by a spectrometric method.
60. The method of claim 59, wherein the spectrometric method comprises fluorescence detection.
61. The method of claim 48, wherein the captured target nucleic acid is from a biological sample.
62. The method of claim 48, wherein the nucleic acid conjugated to the beads is DNA.
63. The method of claim 48, wherein the nucleic acid conjugated to the beads is RNA.
64. A method, comprising:
(a) contacting a target nucleic acid with beads bound to a solid support and further bound to a nucleic acid, wherein target nucleic acid that hybridizes to the nucleic acid bound to the beads is captured, and wherein the solid support is selected from the group consisting of arrays of pits and multiwell supports comprising nanoliter wells; and
(b) detecting captured target nucleic acid.
65. The method of claim 64, wherein the nucleic acid bound to the beads is single-stranded.
66. The method of claim 64, wherein the nucleic acid bound to the beads is DNA.
67. The method of claim 64, wherein the nucleic acid is detected by a spectrometric method.
68. The method of claim 67, wherein the spectrometric method comprises fluorescence detection.
69. A method for capturing a target polynucleotide, which comprises:
contacting a target polynucleotide of a biological sample with a complex comprising a bead conjugated to a solid support and further conjugated to a capture nucleic acid that can hybridize to the target polynucleotide, wherein:
the bead is conjugated to the solid support by an interaction selected from the group consisting of an ionic interaction, polar interaction and hydrophobic interaction; and
the solid support is selected from the group consisting of glass supports, silicon wafers, supports with arrays of pits and supports with nanoliter wells;
whereby the target polynucleotide is captured by the complex.
70. The method of claim 69, wherein the interaction is an ionic interaction.
71. The method of claim 69, wherein the interaction is a polar interaction.
72. The method of claim 69, wherein the interaction is a hydrophobic interaction.
73. The method of claim 69, wherein the solid support is a glass surface.
74. The method of claim 69, wherein the solid support is a support with an array of pits.
75. The method of claim 69, wherein the solid support is a support with nanoliter wells.
76. The method of claim 69, wherein the solid support is a silicon wafer.
77. The method of claim 69, wherein the capture nucleic acid is DNA.
78. The method of claim 69, wherein the capture nucleic acid is RNA.
79. A composition for capturing a target polynucleotide of a biological sample, which comprises a bead of conjugated to a solid suppoer and further conjugated to a capture nucleic acid that can hybridize to the target polynucleotide, wherein:
the bead is conjugated to the solid support by an interaction selected from the group consisting of an ionic interaction, polar interaction and hydrophobic interaction; and
the solid support is selected from the group consisting of glass supports, silicon wafers, supports with arrays of pits and supports with nanoliter wells.
80. The composition of claim 79, wherein the interaction is an ionic interaction.
81. The composition of claim 79, wherein the interaction is a polar interaction.
82. The composition of claim 79, wherein the interaction is a hydrophobic interaction.
83. The composition of claim 79, wherein the solid support is a glass surface.
84. The composition of claim 79, wherein the solid support is a support with an array of pits.
85. The composition of claim 79, wherein the solid support is a support with nanoliter wells.
86. The composition of claim 79, wherein the solid support is a silicon wafer.
87. The composition of claim 79, wherein the nucleic acid bound to the beads is DNA.
88. The composition of claim 19, wherein the nucleic acid bound to the beads is RNA.
This application is a continuation-in-part of U.S. Ser. No. 08/746,036 now U.S. Pat. No. 5,900,481 filed Nov. 6, 1996, entitled “Bead Linkers for Immobilizing Nucleic Acids to Solid Supports”, now U.S. Pat. No. 5,900,481, the teachings of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
In the fields of molecular biology and biochemistry, as well as in the diagnosis of diseases, nucleic acid hybridization has become a powerful tool for the detection, isolation, and analysis of specific oligonucleotide sequences. Typically, such hybridization assays utilize an oligodeoxynucleotide probe that has been immobilized on a solid support; as for example in the reverse dot blot procedure (Saiki, R. K., Walsh, P. S., Levenson, C. H., and Erlich, H. A. (1989) Proc. Natl. Acad Sci. USA 86, 6230). More recently, arrays of immobilized DNA probes attached to a solid surface have been developed for sequencing by hybridization (SBH) (Drmanac, R., Labat, I., Brukner, I., and Crkvenjakov, R. (1989) Genomics, 4, 114-128), (Strezoska, Z., Paunesku, T., Radosavljevic, D., Labat, I., Drmanac, R., and Crkvenjakov, R. (1991) Proc. Natl. Acad. Sci. USA, 88, 10089-10093). SBH uses an ordered array of immobilized oligodeoxynucleotides on a solid support. A sample of unknown DNA is applied to the array, and the hybridization pattern is observed and analyzed to produce many short bits of sequence information simultaneously. An enhanced version of SBH, termed positional SBH (PSBH), has been developed which uses duplex probes containing single-stranded 3′- or 5′-overhangs. (Broude, N. E., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Proc. Natl. Acad Sci. USA, 91, 3072-3076). It is now possible to combine a PSBH capture approach with conventional Sanger sequencing to produce sequencing ladders detectable, for example by gel electrophoresis (Fu, D., Broude, N. E., Koster, H., Smith, C. L., and Cantor, C. R. (1995) Proc. Natl. Acad Sci. USA, 92, 10162-10166)
For the arrays utilized in these schemes, there are a number of criteria which must be met for successful performance. For example, the immobilized DNA must be stable and not desorb during hybridization, washing, or analysis. In addition, the density of the immobilized oligodeoxynucleotide must be sufficient for the ensuing analyses. However, there must be minimal non-specific binding of DNA to the surface. In addition, the immobilization process should not interfere with the ability of immobilized probes to hybridize. For the majority of applications, it is best for only one point of the DNA to be immobilized, ideally a terminus.
In recent years, a number of methods for the covalent immobilization of DNA to solid supports have been developed which attempt to meet all the criteria listed above. For example, appropriately modified DNA has been covalently attached to flat surfaces functionalized with amino acids, (Running, J. A., and Urdea, M. S. (1990) Biotechniques, 8, 276-277), (Newton, C. R., et al., (1993) Nucl. Acids, Res., 21, 1155-1162.), (Nikiforov, T. T., and Rogers, Y. H. (1995) Anal Biochem., 227, 201-209) carboxyl groups, (Zhang, Y., et al., (1991) Nucl. Acids Res., 19, 3929-3933), epoxy groups (Lamture, J. B., et al., (1994) Nucl. Acids Res. 22, 2121-2125), (Eggers, M. D., et al., (1994) BioTechniques, 17, 516-524) or amino groups (Rasmussen, S. R., et al., (1991) Anal. Biochem., 198, 138-142) Although many of these methods were quite successful for their respective applications, when used to link nucleic acids to two-dimensional (flat) supports, the density of the immobilized oligodeoxynucleotide is often insufficient for the ensuing analyses (Lamture, J. B., et al., (1994) Nucl. Acids Res. 22, 2121-2125, Eggers, M. D., et al., (1994) BioTechniques, 17, 516-524).
SUMMARY OF THE INVENTION
In one aspect, the invention features novel compositions comprised of at least one bead conjugated to a solid support and further conjugated to at least one nucleic acid. The bead can be comprised of any of a variety of materials and may be swellable or nonswellable. Preferably the bead is made of a material selected from the group consisting of: silica gel, glass, magnet, Wang resin (4—(hydroxymethyl) phenoxymethylcopoly(styrene—1% divinylbenzene(DVB) resin), metal, plastic, cellulose, dextran cross-linked with epichlorohydrin (e.g., SephadexR), and agarose (e.g., SepharoseR). In a preferred embodiment, the bead is of a size in the range of about 1 to about 100 μm in diameter. In another preferred embodiment, the solid support is selected from the group consisting of: a bead, capillary, plate, membrane, wafer, comb, pin, a wafer with pits, an array of pits or nanoliter wells.
In another aspect, the invention features preferred conjugation means for making the novel compositions. In a preferred embodiment, a covalent amide bond is formed between the bead and the insoluble support In a particularly preferred embodiment, the covalent amide bond is formed by reacting a carboxyl-functionalized bead with an amino-functionalized solid support, or a carboxyl-functionalized support with an amino-functionalized bead.
In a further aspect, the invention features methods for isolating target nucleic acids from a sample or reaction mixture by a conjugation means described herein. In a particularly preferred method, the nucleic acids are directly analyzed by mass spectrometry.
In a final aspect, the invention features kits containing reagents for performing the conjugations and thereby immobilizing nucleic acids to an insoluble support via a bead linker.
As compared to “flat” surfaces, beads linked to a solid support provide an increased surface area for immobilization of nucleic acids. Furthermore, by selecting a bead with the desired functionality, a practitioner can select a functionalization chemistry for immobilizing nucleic acids, which is different from the chemistry of the solid support.
The above and further features and advantages of the instant invention will become clearer from the following Detailed Description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic showing the covalent attachment of a bead to a solid support and DNA to the bead.
FIG. 2 is a schematic showing the covalent attachment of (4—(hydroxymethyl)phenoxymethylcopoly(styrene—1% divinylbenzene(DVB) resin) beads to a solid support as described in Example 1.
FIG. 3 is a schematic representation of nucleic acid immobilization via covalent bifunctional trityl linkers as described in Example 2.
FIG. 4 is a schematic representation of nucleic acid immobilization via hydrophobic trityl linkers as described in Example 3.
FIG. 5 shows a MALDI-TOF mass spectrum of a supernatant of the matrix treated Dynabeads containing bound oligo (5′ iminobiotin-TGCACCTGACTC, SEQ. ID. No. 1). An internal standard (CTGTGGTCGTGC, SEQ. ID. No. 2) was included in the matrix.
FIG. 6 shows a MALDI-TOF mass spectrum of a supernatant of biotin treated Dynabeads containing bound oligo (5′ iminobiotin-TGCACCTGACTC, SEQ. ID. No. 1). An internal standard (CTGTGGTCGTGC, SEQ. ID. No. 2) was included in the matrix.
FIG. 7 schematically depicts conjugation of an unextended primer to a bead via reaction of a 2′, 3′-diol on the primer with boronic acid functionalized beads.
FIG. 8 schematically depicts a pin tool apparatus.
FIG. 9 depicts various pin conformations. FIG. 9A shows a solid pin with a straight head. FIG. 9B shows a solid pin with a concave head. FIG. 9C shows a solid pin with a truncated pyramidal head. FIG. 9D shows a pin with a concave head and a hollowed center (through which can be inserted an optical fibre). FIG. 9E shows a pin with a truncated pyramidal head and a hollowed center.
FIG. 10 is a schematic representation of the conjugation of beads (activated carboxyl) to pins (amino-functionalized) via amide bonds, and attachment of DNA (via an acid-cleavable linker) to beads. A disulfide linker conjugating the beads to the pins and a thioether conjugation between the bead and the trityl group permits selective cleavage of the beads (with DNA still attached) from the pin surface.
FIG. 11 is a schematic representation of paramagnetic beads functionalized with streptavidin to pins via a magnetic interaction and attachment of DNA (via a linker (e.g., modified biotin or photocleavable biotin) to allow selective cleavage of the DNA from the beads.
FIGS. 12A-C schematically represent a pintool apparatus and mount, each separately and a cross section of the mount and tool installed.
FIG. 13 is a schematic representation of mass spectrometry geometries for the pin conformations shown in FIGS. 9A-E.
FIG. 14 schematically depicts a pintool onto which a voltage is applied. When an electrical field is applied, nucleic acids are attracted to the anode. This system purifies nucleic acids, since uncharged molecules would remain in solution, while positively charged molecules are attracted towards the cathode.
FIG. 15 shows a flow chart of the steps involved in sequencing by mass spectrometry using post-biology capture.
DETAILED DESCRIPTION OF THE INVENTION
In general, the invention relates to use of functionalized beads for the immobilization of nucleic acids, wherein the beads are stably associated with a solid support.
FIG. 1 depicts a bead conjugated to a solid support through one or more covalent or non-covalent bonds. Nucleic acids can be immobilized on the functionalized bead before, during or after the bead is conjugated to the solid support. As used herein, the term “nucleic acid” refers to single stranded and/or double stranded polynucleotides such as deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) as well as analogs or derivatives of either RNA or DNA. Also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and the like.
Preferred nucleic acids for use in the subject invention are derivatized to contain at least one reactive moiety. Preferably the reactive moiety is at the 3′ or 5′ end. Alternatively, a nucleic acid can be synthesized with a modified base. In addition, modification of the sugar moiety of a nucleotide at positions other than the 3′ and 5′ position is possible through conventional methods. Also, nucleic acid bases can be modified, e.g., by using N7- or N9-deazapurine nucleosides or by modification of C-5 of dT with a linker arm, e.g., as described in F. Eckstein, ed., “Oligonucleotides and Analogues: A Practical Approach,” IRL Press (1991). Alternatively, backbone-modified nucleic acids (e.g., phosphoroamidate DNA) can be used so that a reactive group can be attached to the nitrogen center provided by the modified phosphate backbone.
In preferred embodiments, modification of a nucleic acid, e.g., as described above, does not substantially impair the ability of the nucleic acid or nucleic acid sequence to hybridize to its complement. Thus, any modification should preferably avoid substantially modifying the functionalities of the nucleic acid which are responsible for Watson-Crick base pairing. The nucleic acid can be modified such that a non-terminal reactive group is present, and the nucleic acid, when immobilized to the support, is capable of self-complementary base pairing to form a “hairpin” structure having a duplex region.
Examples of insoluble supports for use in the instant invention include beads (silica gel, controlled pore glass, magnetic beads, biomagnetic separation beads such as DynabeadsR, Wang resin; Merrifield resin, which is chloromethylated copolystyrene—divinylbenzene(DVB) resin, SephadexR/SepharoseR beads, cellulose beads, etc.), capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold, silver, aluminum, silicon and copper), plastic materials including multiwell plates or membranes (e.g., of polyethylene, polypropylene, polyamide, polyvinylidenedifluoride), wafers, combs, pins or needles (e.g., arrays of pins suitable for combinatorial synthesis or analysis) or beads in an array of pits or nanoliter wells of flat surfaces such as wafers (e.g. silicon wafers), wafers with pits with or without filter bottoms.
An appropriate “bead” for use in the instant invention includes any three dimensional structure that can be conjugated to a solid support and provides an increased surface area for binding of DNA. Preferably the bead is of a size in the range of about 1 to about 100 .mu.m in diameter. For use in the invention, a bead can be made of virtually any insoluble or solid material. For example, the bead can be comprised of silica gel, glass (e.g. controlled-pore glass (CPG)), nylon, Wang resin, Merrifield resin, SephadexR/ SepharoseR, cellulose, magnetic beads, DynabeadsR, a metal surface (e.g. steel, gold, silver, aluminum, silicon and copper), a plastic material (e.g., polyethylene, polypropylene, polyamide, polyester, polyvinylidenedifluoride (PVDF)) and the like. Beads can be swellable, e.g., polymeric beads such as Wang resin, or non-swellable (e.g., CPG).
As used herein, the term “conjugated” refers to ionic or covalent attachment. Preferred conjugation means include: streptavidin- or avidin- to biotin interaction; hydrophobic interaction; magnetic interaction (e.g. using functionalized Dynabeads); polar interactions, such as “wetting” associations between two polar surfaces or between oligo/polyethylene glycol; formation of a covalent bond, such as an amide bond, disulfide bond, thioether bond, or via crosslinking agents; and via an acid-labile linker. In a preferred embodiment for conjugating nucleic acids to beads, the conjugating means introduces a variable spacer between the beads and the nucleic acids. In another preferred embodiment, the conjugation is photocleavable (e.g. streptavidin- or avidin- to biotin interaction can be cleaved by a laser, for example for mass spectrometry).
Appropriate cross-linking agents for use in the invention include a variety of agents that are capable of reacting with a functional group present on a surface of the bead, insoluble support and or nucleic acid and with a functional group present in the nucleic acid and/or bead, respectively. Reagents capable of such reactivity include homo- and hetero-bifunctional reagents, many of which are known in the art. Heterobifunctional reagents are preferred. A preferred bifunctional cross-linking agent is N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB). However, other crosslinking agents, including, without limitation, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-hydrazinonicotimide (HYNIC) may also be used in the novel process. In certain embodiments, the cross-linking agent can be selected to provide a selectively cleavable bond when the nucleic acid molecule is immobilized on the insoluble support. For example, a photolabile cross-linker such as 3-amino-(2-nitrophenyl)propionic acid (Brown et al. (1995) Molecular Diversity 4-12 and Rothschild et al (1996) Nucleic Acids Res. 24:351-66) can be employed to provide a means for cleaving the nucleic acid from. the beads or insoluble (e.g., solid) support, if desired. For further examples of cross-linking reagents, see, e.g., S. S. Wong, “Chemistry of Protein Conjugation and Cross-Linking,” CRC Press (1991), and G. T. Hermanson, “Bioconjugate Techniques,” Academic Press (1995).
In one preferred embodiment, a covalent amide bond is formed between a bead and a insoluble support by reacting a carboxyl-functionalized bead with an amino-functionalized solid support (e.g., as described in Example 1, below, by reacting a carboxyl-functionalized Wang resin with an amino-functionalized silicon surface). Alternatively, a carboxyl-functionalized support can be reacted with an amino-functionalized bead, which take advantage of an acid-cleavable bifunctional trityl protection scheme employed for nucleic acid attachment. The bifunctional trityl linker can also be attached to the 4-nitrophenyl active ester on a resin (e.g. Wang resin) via an amino group as well as from a carboxy group via an amino resin
In the bifunctional trityl approach, the beads may require treatment with a volatile acid (e.g. formic acid, trifluoracetic acid, etc.) to ensure that the nucleic acid is cleaved and can be removed. In which case, the nucleic acid may be deposited as a beadless patch at the bottom of a well in the solid support or on the flat surface of the solid support. After addition of matrix solution, the nucleic acid can then be desorbed into the mass spectrometer, for example.
The hydrophobic trityl linkers can also be exploited as acid-labile linkers by using a volatile acid or an appropriate matrix solution (e.g. a matrix solution containing, for example, 3-hydroxypicolinic acid (3-HPA) to cleave the aminolink trityl group from the nucleic acid molecule). Also, the acid lability can be changed. For example, trityl, monomethoxy, demothoxy- or trimethoxytrityl can be changed to the appropriate p-substituted and even more acid labile tritylamine derivatives of the nucleic acids (i.e. trityl ether and tritylamine bonds to the nucleic acid can be made). Therefore, the nucleic acid may be removed from the hydrophobic linker, for example, by disrupting the hydrophobic attraction or by cleaving tritylether or tritylamine bonds under acidic or the usual mass spectrometry conditions (e.g. wherein the matrix, such as 3-HPA acts as an acid)
As pointed out above, the bead can also be associated with the solid support by non-covalent interactions. For example, a magnetic bead (e.g., a bead capable of being magnetized, e.g., a ferromagnetic bead) can be attracted to a magnetic solid support, and can be released from the support by removal of the magnetic field. Alternatively, the bead can be provided with an ionic or hydrophobic moiety, which can associate with, respectively, an ionic or hydrophobic moiety of the solid support. Also, a bead can be provided with a member of a specific binding pair, and become immobilized to a solid support provided with a complementary binding moiety. For example, a bead coated with avidin or streptavidin can be bound to a surface coated with biotin or derivatives of biotin such as imino-biotin. It will be appreciated that the binding members can be reversed, e.g., a biotin-coated bead can bind to a streptavidin-coated solid support. Other specific binding pairs contemplated for use in the invention include hormone-receptor, enzyme-substrate, nucleic acid-complementary nucleic acid, antibody-antigen and the like.
Examples of preferred binding pairs or linker/interactions are shown in the following Table 1
|streptavidin-biotina, c/photolabile biotinb
||biotinylated pin, avidin beads,
||photolabile biotin DNA
||C18-coated pin, tritylated DNA
||electromagnetic pin, steptavidin
||Dynabeads, biotin DNA
||glass pin, bifunctional trityl-
||silicon wafer, Wang resin,
||silicon wafer, beads are bound
||on the flat surface forming
||arrays or in arrays of nanoliter
||wells, thiol beads, thiolated
||silicon wafer, beads are bound
||on the flat surface forming
||arrays or in arrays of nanoliter
||wells, thiolated DNA
| aThese interactions are reversible
| bThese non-reversible interactions are rapidly cleaved
| cUnless cleavable-linkers are incorporated at some point in the scheme, only the complement of the solid-bound DNA can be analysed in these schemes.
In a particularly preferred embodiment the bead is conjugated to the solid support and/or the nucleic acid is conjugated to the bead using an acid-labile bond. For example, use of a trityl linker, as further described in the following Examples 2 and 3, can provide a covalent or hydrophobic conjugation. Regardless of the nature of the conjugation, the trityl group is readily cleaved in acidic conditions.
A nucleic acid can be bound to a bead which is itself bound to a solid support, e.g., by any of the chemistries discussed above for the attachment of nucleic acids to solid supports, or attachment of beads to solid supports.
In certain embodiments, the invention contemplates the use of orthogonally-cleavable linkers for binding the bead to the solid support, and for binding the nucleic acid to the bead. Thus, a bead can be selectively cleaved from the surface without cleaving the nucleic acid from the bead, while the nucleic acid is cleaved from the bead at a later stage. For example, a disulfide linker (which can be cleaved, using, e.g., DTT) could be employed to bind the bead to the solid surface, and a bead-nucleic acid linker involving an acid-cleavable bifunctional trityl group could be used to immobilize a nucleic acid to the bead. Alternatively the linkage of the nucleic acid could be cleaved while the linkage of the bead to the support remains intact.
A bead can be bound to a solid support through a linking group which can be selected to have a length and a chemical nature such that high-density binding of beads to the solid support, and/or high-density binding of nucleic acid to the beads, is promoted. Such a linking group would have a “tree-like” structure in providing a multiplicity of functional groups per attachment site on the solid support such as polylysine, polyglutamic acid, pentaerythrole and tris-hydroxy-aminomethane.
In certain embodiments, beads can be cross-linked to other beads, e.g., by use of homobifunctional crosslinking reagents. Cross-linked beads can provide additional mechanical strength compared to non-crosslinked beads.
The methods and compositions described herein, can be used to isolate (purify) target nucleic acids from biological samples (reactions). For example, the compositions and methods can be used to isolate particular nucleic acids, which are generated by cloning (Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989), polymerase chain reaction (PCR) (C. R. Newton and A. Graham, PCR, BIOS Publishers, 1994), ligase chain reaction (LCR) (Wiedmann, M., et al., (1994) PCR Methods Appl. Vol. 3, Pp. 57-64; F. Barany Proc. Natl. Acad. Sci USA 88, 189-93 (1991), strand displacement amplification (SDA) (G. Terrance Walker et al., Nucleic Acids Res. 22, 2670-77 (1994)) European Patent Publication Number 0 684 315 entitled “Strand Displacement Amplification Using Thermophilic Enzymes”) and variations such as RT-PCR (Higuchi, et al., Bio/Technology 11:1026-1030 (1993)), allele-specific amplification (ASA), cycle sequencing and transcription based processes.
Further, the methods and compositions can be used to isolate or transfer particular nucleic acids during the performance of a particular reaction. For example, a PCR reaction can be performed to ‘master’ mix without addition of the dideoxynucleotides (d/ddNTPs) or sequencing primers. Aliquots can then be isolated via a conjugation means described herein and transferred, for example to a sequencing plate, where d/ddNTPs and primers can then be added to perform a sequencing reaction. Alternatively, the PCR can be split between A, C, G, and T master mixes. Aliquots can then be transferred to a sequencing plate and sequencing primers added.
For example, 0.4-0.5 pmol of PCR product can be used in a cycle-sequencing reaction using standard conditions, allowing each PCR to be used for 10 sequencing reactions (10×A, C, G, and T). The sequencing reactions can be carried out in a volume of 10 μl containing 5-6 pmol of 5′-labeled sequencing primer in a standard 384 microwell plate allowing up to 96 sequencing reactions (3360 bases at 35 bases per reaction). Alternatively, a 192 microwell plate approximately 5×5 cm in a 12×16 format can be used. This format allows up to 48 sequencing reactions to be carried out per well, resulting in 1680 bases per plate (at 35 bases per reaction). The format of the sequencing plate will determine the dimensions of the transfer agent (e.g. pin-tool).
A pin tool in a 4×4 array (FIG. 8) can be applied to the wells of the sequencing plate and the sequencing products captured on functionalized beads as described herein, which are attached to the tips of the pins (>=1 pmol capacity). During the capture/incubation step, the pins can be kept in motion (vertical, 1-2 mm travel) to mix the sequencing reaction and increase the efficiency of the capture.
Alternatively, the nucleic acid can be directly captured onto the pin-tool, for example, a linking functionality on the pin-tool can immobilize the nucleic acid upon contact. Further, immobilization can result from application to the pin-tool of an electrical field, as shown in FIG. 14. When a voltage is applied to the pin-tool, the nucleic acids are attracted to the anode. This system also purifies nucleic acids, since uncharged molecules remain in solution and positively charged molecules are attracted to the cathode. For more specificity, the pin-tool (with or without voltage), can be modified to contain a partially or fully single stranded oligonucleotide (e.g. about 5-12 base pairs). Only complementary nucleic acid sequences (e.g. in solution) are then specifically conjugated to the pins.
In yet a further embodiment, a PCR primer can be conjugated to the tip of a pin-tool. PCR can be performed with the solid phase (pin-tool)-bound primer and a primer in solution, so that the PCR product becomes attached to the pin-tool. The pin-tool with the amplification product can then be removed from the reaction and analyzed (e.g. by mass spectrometry).
Examples of different pin conformations are shown in FIG. 9. For example, FIGS. 9a, 9b. and 9c. show a solid pin configuration. FIGS. 9d. and 9e show pins with a channel or hole through the center, for example to accomodate an optic fibre for mass spectrometer detection. The pin can have a flat tip or any of a number of configurations, including nanowell, concave, convex, truncated conic or truncated pyramidal (e.g. size 4-800μ across ×100μ depth). In a preferred embodiment, the individual pins are about 5 mm in length and about 1 mm in diameter. The pins and mounting plate can be made of polystyrene (e.g. one-piece injection moulded). Polystyrene is an ideal material to be functionalised and can be moulded with very high tolerances. The pins in a pin-tool apparatus may be collapsible (eg, controlled by a scissor-like mechanism), so that pins may be brought into closer proximity, reducing the overall size
Captured nucleic acids can be analyzed by any of a variety of means including, for example, spectrometric techniques such as UV/VIS, IR, fluorescence, chemiluminescence, or NMR spectroscopy, mass spectrometry, or other methods known in the art, or combinations thereof. Preferred mass spectrometer formats include ionization (I) techniques, such as matrix assisted laser desorption (MALDI), continuous or pulsed electrospray (ESI) and related methods (e.g. Ionspray or Thermospray), or massive cluster impact (MCI); these ion sources can be matched with detection formats including linear or non-linear reflectron time-of-flight (TOF), single or multiple quadrupole, single or multiple magnetic sector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, and combinations thereof (e.g., ion-trap/time-of-flight). For ionization, numerous matrix/wavelength combinations (MALDI) or solvent combinations (ESI) can be employed.
If conditions preclude direct analysis of captured DNA, then the DNA can be released and/or transferred. However, it may be important that the advantages of sample concentration are not lost at this stage. Ideally, the sample should be removed from the surface in as little a volume of eluent as possible, and without any loss of sample. Another alternative is to remove the beads (+sample) from the surface, where relevant, and measure the sample directly from the beads.
For example, for detection by mass spectrometry, the pin-tool can be withdrawn and washed several times, for example in ammonium citrate to condition the sample before addition of matrix. For example, the pins can simply be dipped into matrix solution. The concentration of matrix can then be adjusted such that matrix solution only adheres to the very tip of the pin. Alternatively, the pintool can be inverted and the matrix solution sprayed onto the tip of each pin by a microdrop device. Further, the products can be cleaved from the pins, for example into a nanowell on a chip, prior to addition of matrix.
For analysis directly from the pins, a stainless steel ‘mask’ probe can be fitted over the pins in one scheme (FIG. 12) which can then be installed in the mass spectrometer.
Two mass spectrometer geometries for accomodating the pin-tool apparatus are proposed in FIG. 13. The first accomodates solid pins. In effect, the laser ablates a layer of material from the surface of the crystals, the resultant ions being accelerated and focused through the ion optics. The second geometry accomodates fibre optic pins in which the samples are lasered from behind. In effect, the laser is focused onto the pin-tool back plate and into a short optical fibre (about 100 μm in diameter. and about 7 mm length to include thickness of the back plate). This geometry requires the volatilised sample to go through the depth of the matrix/bead mix, slowing and cooling down the ions resulting in a type of delayed extraction which should actually increase the resolution of the analysis.
The probe through which the pins are fitted can also be of various geometries. For example, a large probe with multiple holes, one for each pin, fitted over the pin-tool. The entire assembly is translated in the X-Y axes in the mass spectrometer. Alternatively, as a fixed probe with a single hole, which is large enough to give an adequate electric field, but small enough to fit between the pins. The pin-tool is then translated in all three axes with each pin being introduced through the hole for sequential analyses This format is more suitable for the large pin-tool (i.e. based on a standard 384 well microplate format). The two probes described above, are both suitable for the two mass spectrometer geometries described above.
FIG. 15 schematically depicts the steps involved in mass spectrometry sequencing by post biology capture as described above.
The methods of the invention are useful for providing spatially-addressable arrays of nucleic acids immobilized on beads, which are further attached to solid supports. Such spatially addressable or pre-addressable arrays are useful in a variety of processes (e.g., SBH, quality control, and DNA sequencing diagnostics). In another aspect, the invention provides combinatorial libraries of immobilized nucleic acids bound to beads, which are further bound to a solid support as described above.
In still another aspect, the invention provides a kit for immobilizing nucleic acids on beads, which are further bound to a solid support. In one embodiment, the kit comprises an appropriate amount of: i) beads, and/or ii) the insoluble support, and iii) conjugation means. The kits described herein can also optionally include appropriate buffers; containers for holding the reagents; and/or instructions for use.
The present invention is further illustrated by the following Examples, which are intended merely to further illustrate and should not be construed as limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
EXAMPLE 1 Attachment of Resin Beads to a Silicon Surface
A silicon surface (e.g. of a silicon wafer) is derivatized with amino groups by treatment with 3-aminopropyltriethoxysilane. Wang resin beads are treated with succinic anhydride to provide carboxyl-functionalized resin beads. The carboxyl-functionalized resin beads are then coupled to the amino-functionalized silicon surface with a coupling reagent (for example, dicyclohexylcarbodiimide (DCC)), in the presence of p-nitrophenol. The resin beads become covalently linked to the silicon surface, and the unreacted carboxyl groups of the resin are converted to the p-nitrophenyl ester (an activated ester suitable for coupling with a nucleic acid).
Alternatively, the carboxyl groups of the Wang resin are transformed to the p-nitrophenyl active esters prior to reacting with the amino-functionalized silicon surface.
Thus, resin beads can be rapidly and conveniently attached to a silicon surface, and can be simultaneously converted to a reactive form suitable for covalent attachment of nucleic acids.
EXAMPLE 2 Immobilization of Nucleic Acids on Solid Supports via an Acid-labile Covalent Bifunctional Trityl Linker
Aminolink DNA was prepared and purified according to standard methods. A portion (10 eq) was evaporated to dryness on a speedvac and suspended in anhydrous DMF/pyridine (9:1; 0.1 ml). To this was added the chlorotrityl chloride resin (1 eq, 1.05 mol/mg loading) and the mixture was shaken for 24 hours. The loading was checked by taking a sample of the resin, detritylating this using 80% AcOH, and measuring the absorbance at 260nm. Loading was ca. 150 pmol/mg resin.
In 80% acetic acid, the half-life of cleavage was found to be substantially less than 5 minutes—this compares with trityl ether-based approaches of half-lives of 105 and 39 minutes for para and meta substituted bifunctional dimethoxytrityl linkers respectively. Preliminary results have also indicated that the 3-hydroxy picolinic acid matrix alone is sufficient to cleave the DNA from the chlorotrityl resin during MALDI mass spectrometry.
EXAMPLE 3 Immobilization of Nucleic Acids on Solid Supports via Hydrophobic Trityl Linker
The primer contained a 5′-dimethoxytrityl group attached using routine trityl-on DNA synthesis.
C18 beads from an oligo purification cartridge (0.2 mg) placed in a filter tip was washed with acetonitrile, then the solution of DNA (50 ng in 25 l) was flushed through. This was then washed with 5% acetonitrile in ammonium citrate buffer (70 mM, 250 l). To remove the DNA from the C18, the beads were washed with 40% acetonitrile in water (10 l) and concentrated to ca 2 l on the Speedvac or directly subjected to MALDI mass spectrometry.
Alternatively C18 beads were first covalently attached to a silicon surface (e.g. a silicon wafer) or adsorbed to a flat surface by hydrophobic interaction.
The results showed that acetonitrile/water at levels of ca.>30% are enough to dissociate the hydrophobic interaction. Since the matrix used in MALDI contains 50% acetonitrile, the DNA can be released from the support and MALDIed successfully (with the trityl group removed during the MALDI process)
EXAMPLE 4 Attaching Beads to Silicon Chips
Amino derivatisation of silicon surface
The silicon wafers were washed with ethanol to remove surface debris and flamed over a bunsen burner until “red hot” to ensure oxidation of the surface. After cooling, the wafers were immersed in an anhydrous solution of 3-aminopropyltriethoxysilane in toluene (25%v/v) for 3 hours. The wafers were then washed with toluene (three times) then anhydrous dimethylacetamide (three times).
Activation of Wang resin beads
Vacuum-dried Wang resin beads (5g, 0.84mmol/g loading, 4.2 mmol, diameter 100-200 mesh), obtained from Novabiochem, were suspended in pyridine (40 ml) with DMAP (0.1 eq, 0.42 mmol, 51 mg). To this was added succinic anhydride (5 eq, 21 mmol, 2.10 g) and the reaction was shaken for 12 hours at room temperature. After this time, the beads were washed with dimethylformamide (three times), then pyridine (three times) and suspended in pyridine/dimethylformamide (1:1, 20 ml). 4-Nitrophenol (2 eq, 8.4 mmol, 1.40 g) was added and the condensation was activated by adding dicyclohexylcarbodiimide (DCC) (2 eq, 8.4 mmol, 1.73 g) and the reaction mixture was shaken for 12 hours. The beads were then washed with dimethylformamide, pyridine and hexane, and stored at 4° C.
Coupling of Beads to Silicon Wafers
The amino-derivatised silicon wafer is treated with a suspension of the 4-nitrophenol beads in dimethyl acetamide (DMA), and within five minutes, the beads are covalently linked to the surface. The coated surface can then be washed with DMA, ethanol and water, under which conditions the beads remain as a uniform monolayer. Care must be taken to avoid scratching the beaded surface. The beads can then be reacted with the amino-functionalised modified DNA.
EXAMPLE 5 Immobilization of Nucleic Acids on Solid Supports via Streptavidin-Iminobiotin
2-iminobiotin N-hydroxy-succinimid ester (Sigma) was conjugated to the oligonucleotides with a 3′- or 5′-amino linker following the conditions suggested by the manufacture. The completion of the reaction was confirmed by MALDI-TOF MS analysis and the product was purified by reverse phase HPLC.
For each reaction, 0.1 mg of streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin from Dynal) were incubated with 80 pmol of the corresponding oligo in the presence of 1M NaCl and 50 mM ammonium carbonate (pH 9.5) at room temperature for one hour. The beads with bound oligonucleotides were washed twice with 50 mM ammonium carbonate (pH 9.5) Then the beads were incubated in 2 μl of 3-HPA matrix at room temperature for 2 min. An aliquot of 0.5 μl of supernatant was applied to MALDI-TOF. For biotin displacement experiment, 1.6 nmol of free biotin (80 fold excess to the bound oligo) in 1 μl of 50 mM ammonium citrate was added to the beads. After a 5 min. incubation at room temperature, 1 μl of 3-HPA matrix was added and 0.5 μl of supernatant was applied to the MALDI-TOF MS. To maximize the recovery of the bound iminobiotin oligo, the beads from the above treatment were again incubated with 2 μl of 3-HPA matrix and 0.5 μl of the supernatant was applied to MALDI-TOF MS.
Both matrix alone and free biotin treatment quantitatively released iminobiotin oligo off the streptavidin beads as shown in FIGS. 5 and 6. Almost no bound oligo was observed after the second treatment which confirmed the complete recovery
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.
| Patente citada|| Fecha de presentación|| Fecha de publicación|| Solicitante|| Título|
|US4139346||28 Nov 1977||13 Feb 1979||Enzo Bio Chem Incorporated||Nucleic acid and protein binding paper|
|US4582789||18 Dic 1984||15 Abr 1986||Cetus Corporation||Containing alkylating intercalation moiety|
|US4683194||28 Mar 1985||28 Jul 1987||Cetus Corporation||Method for detection of polymorphic restriction sites and nucleic acid sequences|
|US4683195||7 Feb 1986||28 Jul 1987||Cetus Corporation||One of the first Polymerase Chain Reaction (PCR) patents|
|US4725677||10 Ago 1984||16 Feb 1988||Biosyntech Gmbh||Process for the preparation of oligonucleotides|
|US4729947||29 Mar 1984||8 Mar 1988||The Board Of Regents Of The University Of Nebraska||DNA sequencing|
|US4749742||18 Jul 1986||7 Jun 1988||The Queens's University Of Belfast||Solid phase peptide synthesis|
|US4757141||26 Ago 1985||12 Jul 1988||Applied Biosystems, Incorporated||Conjugates with oligonucleotides|
|US4794150||11 Mar 1987||27 Dic 1988||Samuel Steel||Synthesis of peptide analogs|
|US4797355||13 Jun 1985||10 Ene 1989||Amgen Inc.||Methods for attaching polynucleotides to supports|
|US4806546||30 Sep 1985||21 Feb 1989||Miles Inc.||Immobilization of nucleic acids on derivatized nylon supports|
|US4855225||7 Feb 1986||8 Ago 1989||Applied Biosystems, Inc.||Method of detecting electrophoretically separated oligonucleotides|
|US4882127||12 Nov 1987||21 Nov 1989||Akademie Der Wissenschaften Der Ddr||Made up of sequencing blocks with lids, sample dosing system, holder for carrier matrix and punching device|
|US4948882||4 May 1987||14 Ago 1990||Syngene, Inc.||Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis|
|US4983521||12 Sep 1986||8 Ene 1991||The Regents Of The University Of California||Transmembrane integrator sequences|
|US4994373||20 Jul 1989||19 Feb 1991||Enzo Biochem, Inc.||Method and structures employing chemically-labelled polynucleotide probes|
|US5003059||20 Jun 1988||26 Mar 1991||Genomyx, Inc.||Determining DNA sequences by mass spectrometry|
|US5037882||23 Dic 1988||6 Ago 1991||Steel Samuel L||Synthesis of oligonucleotide analogs|
|US5045694||27 Sep 1989||3 Sep 1991||The Rockefeller University||Instrument and method for the laser desorption of ions in mass spectrometry|
|US5064754||13 Nov 1987||12 Nov 1991||Mills Randell L||Generating specific polynucleotides, determining mucleic acid base composition and terminal base, solving the base seuence u sing an logorithm called the matrix method of analysis|
|US5077210||13 Ene 1989||31 Dic 1991||Eigler Frances S||Immobilization of active agents on substrates with a silane and heterobifunctional crosslinking agent|
|US5082935||15 Dic 1988||21 Ene 1992||Amoco Corporation||For detecting RNA or DNA|
|US5118605||29 Sep 1988||2 Jun 1992||Chiron Corporation||Polynucleotide determination with selectable cleavage sites|
|US5118937||21 Ago 1990||2 Jun 1992||Finnigan Mat Gmbh||Process and device for the laser desorption of an analyte molecular ions, especially of biomolecules|
|US5135870||1 Jun 1990||4 Ago 1992||Arizona Board Of Regents||Laser ablation/ionizaton and mass spectrometric analysis of massive polymers|
|US5143854||7 Mar 1990||1 Sep 1992||Affymax Technologies N.V.||Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof|
|US5149625||28 Mar 1990||22 Sep 1992||President And Fellows Of Harvard College||Sequencing|
|US5198531||14 Jun 1991||30 Mar 1993||Research Diagnostic Antibodies||Polymeric resin for peptide synthesis|
|US5210412||31 Ene 1991||11 May 1993||Wayne State University||Method for analyzing an organic sample|
|US5221518||13 Ago 1991||22 Jun 1993||Mills Randell L||DNA sequencing apparatus|
|US5234824||2 Jun 1992||10 Ago 1993||Specialty Laboratories, Inc.||Gently lysing blood cells with solution of detergent and viscosity-increasing agent, trapping DNA on filter; recovering in water, analyzing|
|US5237016||5 Ene 1989||17 Ago 1993||Siska Diagnostics, Inc.||End-attachment of oligonucleotides to polyacrylamide solid supports for capture and detection of nucleic acids|
|US5242974||22 Nov 1991||7 Sep 1993||Affymax Technologies N.V.||Polymer reversal on solid surfaces|
|US5283342||9 Jun 1992||1 Feb 1994||Neorx Corporation||Complexes to enhance delivery of tumor drugs|
|US5288644||13 Nov 1992||22 Feb 1994||The Rockefeller University||High speed, automatic detection of mass weights of DNA fragments; mass spectrometry|
|US5380489||18 Feb 1992||10 Ene 1995||Eastman Kodak Company||Element and method for nucleic acid amplification and detection using adhered probes|
|US5380833||13 Dic 1991||10 Ene 1995||Chiron Corporation||Polynucleotide reagents containing selectable cleavage sites|
|US5410068||23 Oct 1989||25 Abr 1995||Perseptive Biosystems, Inc.||Succinimidyl trityl compounds and a process for preparing same|
|US5430136||27 Jul 1990||4 Jul 1995||Chiron Corporation||Oligonucleotides having selectably cleavable and/or abasic sites|
|US5436327||21 Sep 1989||25 Jul 1995||Isis Innovation Limited||Support-bound oligonucleotides|
|US5474895||14 Nov 1991||12 Dic 1995||Siska Diagnostics Inc.||Non-isotopic detection of nucleic acids using a polystyrene support-based sandwich hybridization assay and compositions useful therefor|
|US5478893||5 Ago 1993||26 Dic 1995||Siska Diagnostics Inc.||End-attachment of oligonucleotides to polyacrylamide solid supports for capture and detection of nucleic acids|
|US5484701||31 Ene 1992||16 Ene 1996||E. I. Du Pont De Nemours And Company||Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products|
|US5492821||13 Nov 1991||20 Feb 1996||Cargill, Inc.||Diagnostics, food industry|
|US5503980||17 Oct 1994||2 Abr 1996||Trustees Of Boston University||Determining nucleotide sequence of nucleic acid target using nucleic acid probes|
|US5506348||24 Feb 1994||9 Abr 1996||Ciba-Geigy Corporation||Composition containing ammonium salt of organic or inorganic acid, hydroxy substituted phenone, high molecular weight compound|
|US5512439 *||6 Jul 1994||30 Abr 1996||Dynal As||Oligonucleotide-linked magnetic particles and uses thereof|
|US5514548||17 Feb 1994||7 May 1996||Morphosys Gesellschaft Fur Proteinoptimerung Mbh||Method for in vivo selection of ligand-binding proteins|
|US5516635||15 Oct 1992||14 May 1996||Ekins; Roger P.||Binding assay employing labelled reagent|
|US5527675||20 Ago 1993||18 Jun 1996||Millipore Corporation||Method for degradation and sequencing of polymers which sequentially eliminate terminal residues|
|US5541313||9 Nov 1994||30 Jul 1996||Molecular Biosystems, Inc.||Single-stranded labelled oligonucleotides of preselected sequence|
|US5545539||18 Oct 1994||13 Ago 1996||Genzyme Corporation||Plymerase chain reaction, n,n,n-trimethylglycine used as amplifier agent, target trinucleotide repeat sequence, indicative or huntington's disease|
|US5547835||6 Ene 1994||20 Ago 1996||Sequenom, Inc.||Using the sanger sequencing strategy and assembling the sequence information by analysis of the nested fragments obtained by base-specific chain termination via their different molecular masses|
|US5552535||7 Nov 1994||3 Sep 1996||Zeneca Limited||Multiple oligonucleotide containing oligomers and the cleanable linkers used in their preparation|
|US5571902||26 May 1994||5 Nov 1996||Isis Pharmaceuticals, Inc.||Synthesis of oligonucleotides|
|US5580733||6 Sep 1994||3 Dic 1996||Wayne State University||Illumination mixture of molecule on matrix using laser|
|US5583042||22 Mar 1994||10 Dic 1996||Neose Pharmaceuticals, Inc.||Binary mixtures containing glycosyltransferases|
|US5601982||7 Feb 1995||11 Feb 1997||Sargent; Jeannine P.||Method and apparatus for determining the sequence of polynucleotides|
|US5604097||19 Dic 1994||18 Feb 1997||Spectragen, Inc.||Methods for sorting polynucleotides using oligonucleotide tags|
|US5605798||17 Mar 1995||25 Feb 1997||Sequenom, Inc.||Detecting target nucleic acid sequences|
|US5612474||30 Jun 1994||18 Mar 1997||Eli Lilly And Company||Acid labile immunoconjugate intermediates|
|US5616698||10 Ene 1994||1 Abr 1997||University Of Toronto Innovations Foundation||Reacting a saccharide having atleast one monosaccharide unit having hydroxyl group with polyoxyethylene glycol which is end capped both ends with methanol and a diol to form ether glycoside linkage, activation, glycosylation and capping|
|US5616700||18 Nov 1993||1 Abr 1997||Beckman Instruments, Inc.||Processes for synthesizing nucleotides and modified nucleotides using N.sub.|
|US5622824||10 Feb 1995||22 Abr 1997||Sequenom, Inc.||DNA sequencing by mass spectrometry via exonuclease degradation|
|US5624711||27 Abr 1995||29 Abr 1997||Affymax Technologies, N.V.||Derivatization of solid supports and methods for oligomer synthesis|
|US5631134||5 Jun 1995||20 May 1997||The Trustees Of Boston University||Methods of preparing probe array by hybridation|
|US5635598||21 Jun 1994||3 Jun 1997||Selectide Corporation||Selectively cleavabe linners based on iminodiacetic acid esters for solid phase peptide synthesis|
|US5639633||6 Jun 1995||17 Jun 1997||Cargill, Incorporated||Stabilizing the protein by biochemical conjugation with linker groups|
|US5641862||29 Mar 1995||24 Jun 1997||The Regents Of The University Of California||General method for producing and selecting peptides with specific properties|
|US5643722||11 May 1994||1 Jul 1997||Trustees Of Boston University||Marking proteins and detection, incubation|
|US5643798||7 Jun 1995||1 Jul 1997||The Rockefeller University||Instrument and method for the sequencing of genome|
|US5648462||27 Ene 1995||15 Jul 1997||Setsuko Funakoshi||Peptide purification method using novel linker and solid-phase ligand|
|US5648480||6 Jun 1995||15 Jul 1997||Northwestern University||Process for making oligonucleotides having modified internucleoside linkages|
|US5650277||4 Oct 1994||22 Jul 1997||Diagenetics Ltd.||Method of determining the presence and quantifying the number of di- and trinucleotide repeats|
|US5652358||3 Nov 1994||29 Jul 1997||Hoechst Aktiengesellschaft||Solid-phase synthesis of oligoribonucleotides|
|US5663242||31 Mar 1995||2 Sep 1997||Siska Diagnostics, Inc.||End-attachment of oligonucleotides to polyacrylamide solid supports for capture and detection of nucleic acids|
|US5668266||12 May 1995||16 Sep 1997||Syngene, Inc.||Single stranded oligonucleotides|
|US5670322||1 Jun 1995||23 Sep 1997||Houston Advanced Res Center||Targets, electronic signals and detection of frequency dependent electrical signals|
|US5677195||20 Nov 1992||14 Oct 1997||Affymax Technologies N.V.||Forming oligonucleotides or peptides by forming flow channels in substrate surface, each of flow channels comprising walls which form fluid-tight seals with surface and a flow intake and outlet, flowing nucleic acids through channels to bind|
|US5679773||17 Ene 1995||21 Oct 1997||Affymax Technologies N.V||Reagants and methods for immobilized polymer synthesis and display|
|US5688642||1 Dic 1994||18 Nov 1997||The United States Of America As Represented By The Secretary Of The Navy||Selective attachment of nucleic acid molecules to patterned self-assembled surfaces|
|US5691141||6 Jun 1995||25 Nov 1997||Sequenom, Inc.||DNA sequencing by mass spectrometry|
|US5700642||22 May 1995||23 Dic 1997||Sri International||Oligonucleotide sizing using immobilized cleavable primers|
|US5726243||3 Jul 1996||10 Mar 1998||Regents Of The University Of Minnesota||Mild solid-phase synthesis of aligned, branched triple-helical peptides|
|US5736625||6 Jun 1995||7 Abr 1998||Cargill, Incorporated||Method for stabilizing proteins with saccharide linked protein polymer conjugates|
|US5736626||29 Ene 1996||7 Abr 1998||The Perkin-Elmer Corporation||Diglycolate synthesis supports|
|US5742049||20 Mar 1996||21 Abr 1998||Bruker-Franzen Analytik Gmbh||Method of improving mass resolution in time-of-flight mass spectrometry|
|US5795714||23 Ago 1993||18 Ago 1998||Trustees Of Boston University||Immobilization; hybridization|
|US5830655||26 Abr 1996||3 Nov 1998||Sri International||Oligonucleotide sizing using cleavable primers|
|US5864137||1 Oct 1996||26 Ene 1999||Genetrace Systems, Inc.||Mass spectrometer|
|US5869242||18 Sep 1995||9 Feb 1999||Myriad Genetics, Inc.||Rapid nonsequencing analysis of denatured nucleic acid segments for determination of mutations by comparision to wild-type and known variations; for genetic screening and diagnosis|
|US5900481 *||6 Nov 1996||4 May 1999||Sequenom, Inc.||Bead linkers for immobilizing nucleic acids to solid supports|
|US6376044||31 May 2000||23 Abr 2002||Waters Investments Limited||Enhanced resolution matrix-laser desorption and ionization TOF-MS sample surface|
|DE3930312A1 *||11 Sep 1989||26 Abr 1990||Max Planck Gesellschaft||Verfahren zur direkten sequenzierung von nukleinsaeureketten|
|DE4011991A1 *||12 Abr 1990||18 Oct 1990||Hitachi Ltd||Simultaneous sequencing of several DNA samples - by cloning into separate vectors, complementary strand synthesis from specific fluorescent labelled primers, electrophoretic sepn. etc.|
|EP0054450A1 *||10 Nov 1981||23 Jun 1982||Jean-Claude Lafon||Hearing aid devices|
|EP0217403A2||3 Oct 1986||8 Abr 1987||Abbott Laboratories||Solid-phase analytical device and method for using same|
|EP0253578A2||9 Jul 1987||20 Ene 1988||Hybritech Incorporated||A solid phase system incorporating tetrazolium salts for use in ligand-receptor assays|
|EP0264036A2||2 Oct 1987||20 Abr 1988||Abbott Laboratories||Analytical device and method for detecting chlamydia trachomatis and neisseria gonorrhoeae|
|EP0264036B1||2 Oct 1987||30 Jun 1993||Abbott Laboratories||Analytical device and method for detecting chlamydia trachomatis and neisseria gonorrhoeae|
|EP0360677A1||18 Sep 1989||28 Mar 1990||Commissariat A L'energie Atomique||Method and equipment for the identification of DNA bases|
|EP0420053A1||21 Sep 1990||3 Abr 1991||W.R. Grace & Co.-Conn.||Improved solid assay support systems|
|EP0684315A1 *||13 Mar 1995||29 Nov 1995||Becton Dickinson and Company||Strand displacement amplification using thermophilic enzymes|
|EP0701001A2 *||7 Sep 1995||13 Mar 1996||Hitachi, Ltd.||DNA separating, fractionating and analyzing method and system therefor|
|GB2017105A *|| ||Título no disponible|
|JPH02215399A *|| ||Título no disponible|
|JPH06294796A *|| ||Título no disponible|
|JPS63230086A *|| ||Título no disponible|
|WO1984002579A1 *||21 Dic 1983||5 Jul 1984||Cooper Lipotech Inc||Lipid-vesicle-surface assay reagent and method|
|WO1989009282A1 *||22 Mar 1989||5 Oct 1989||Cemu Bioteknik||Method of sequencing dna|
|WO1989009406A1 *||28 Mar 1989||5 Oct 1989||Uwe B Sleytr||Process for immobilizing or depositing molecules or substances on a support|
|WO1989012624A2 *||12 Jun 1989||28 Dic 1989||Cetus Corp||Coupling agents and sterically hindered disulfide linked conjugates prepared therefrom|
|WO1990001564A1 *||7 Ago 1989||22 Feb 1990||Microprobe Corp||Methods for multiple target analyses through nucleic acid hybridization|
|WO1990003382A1 *||21 Sep 1989||5 Abr 1990||Isis Innovation||Support-bound oligonucleotides|
|WO1990007582A1 *||4 Ene 1990||6 Jul 1990||Siska Diagnostics Inc||End-attachment of oligonucleotides to polyacrylamide solid supports for capture and detection of nucleic acids|
|WO1990015883A1 *||30 May 1990||15 Dic 1990||Univ Utah||Dna sequencing using low fluorescence background electroblotting membrane|
|WO1991006678A1 *||26 Oct 1990||27 Abr 1991||Stanford Res Inst Int||Dna sequencing|
|WO1991013075A2 *||15 Feb 1991||5 Sep 1991||Orion Yhtymae Oy||Method and reagent for determining specific nucleotide variations|
|WO1992003575A1 *||14 Ago 1991||5 Mar 1992||Medical Res Council||Method for preparing, isolating and sequencing polynucleotides|
|WO1992007879A1 *||22 Oct 1991||6 May 1992||Minnesota Mining & Mfg||Biological active material covalently immobilized on to azlactone-functional polymeric supports and method for preparing it|
|WO1992010092A1 *||20 Nov 1991||7 Jun 1992||Affymax Tech Nv||Very large scale immobilized polymer synthesis|
|WO1992013629A1 *||30 Ene 1992||20 Ago 1992||Univ Wayne State||A method for analyzing an organic sample|
|WO1992015712A1 *||4 Mar 1992||17 Sep 1992||Molecular Tool Inc||Nucleic acid typing by polymerase extension of oligonucleotides using terminator mixtures|
|WO1993006925A1 *||2 Oct 1992||15 Abr 1993||Minnesota Mining & Mfg||Covalently reactive particles incorporated in a continuous porous matrix|
|WO1993009668A1 *||20 Nov 1992||27 May 1993||Affymax Tech Nv||Combinatorial strategies for polymer synthesis|
|WO1993020236A1 *||31 Mar 1993||14 Oct 1993||Applied Biosystems||Probe composition and method|
|WO1994011421A1 *||5 Nov 1993||26 May 1994||Pharmacia Lkb Biotech||A method of surface modification|
|WO1994011529A1 *||5 Nov 1993||26 May 1994||Ulf Landegren||A method of processing nucleic acid samples|
|WO1994011530A1 *||5 Nov 1993||26 May 1994||Univ Boston||Positional sequencing by hybridization|
|WO1994011735A1 *||28 Oct 1993||26 May 1994||Erkki Soini||Method and apparatus for bioaffinity assays|
|WO1994016101A2 *||6 Ene 1994||21 Jul 1994||Hubert Koester||Dna sequencing by mass spectrometry|
|WO1994021822A1 *||18 Mar 1994||29 Sep 1994||Hubert Koester||Dna sequencing by mass spectrometry via exonuclease degradation|
|WO1995004524A1 *||8 Jul 1994||16 Feb 1995||Yechezkel Barenholz||A method for high loading of vesicles with biopolymeric substances|
|WO1995030773A1 *||5 May 1995||16 Nov 1995||Allan Asp||Method of nucleic acid transfer|
|WO1995031429A1 *||11 May 1995||23 Nov 1995||Univ Boston||Photocleavable agents and conjugates for the detection and isolation of biomolecules|
|WO1996037630A1 *||30 Abr 1996||28 Nov 1996||Stanford Res Inst Int||Oligonucleotide sizing using cleavable primers|
|1||Albretsen et al., "Applications of Magnetic Beads with Covalently Attached Oligonucleotides in Hybridization: Isolation and Detection of Specific Measles Virus mRNA from a Crude Cell Lysate", Anal. Biochem., 189:40-50, 1990.|
|2||Alderton et al., Magnetic bead purification of M13 DNA sequencing templates, Anal. Biochem. 201:166-169 (1992).|
|3||Allin, S.M.and Shuttleworth, S.J., "The Preparation and First Application of a Polymer Supported "Evans" Oxazolidinone", Tetrahedron Lett., 37(44):8023-8026 (1996).|
|4||Arlinghaus et al., Applications of resonance ionization spectroscopy for semiconductor, environmental and biomedical analysis, and for DNA sequencing, SPIE, vol. 1435, Opt. Methods Ultrasensitive Detect. Anal. Tech. Appl. pp. 26-35 (1991).|
|5||Arshady, R, Beaded polymer supports and gels: 1. Manufacturing techniques, Journal of Chromatography, 586:181-197 (1991).|
|6||Arshady, R., Beaded polymer supports and gels: 11. Physico-chemical criteria and functionalization, Journal of Chromatography, 586:199-219 (1991).|
|7||Arshady, Reza, Beaded Polymer Supports and Gels, I. Manufacturing Techniques, Journal of Chromatography, (1991), pp. 181-197, vol. 586, No. 23, Elsevier Science Publishers, B.V., Amsterdam.|
|8||Arshady, Reza, Beaded Polymer Supports and Gels, II. Physico-chemical Criteria and Functionalization, Journal of Chromatography, (1991), pp. 199-219, vol. 586, No. 23, Elsevier Science Publishers, B.V. Amsterdam.|
|9||Backes, B. J. et al., "Activation Method to Prepare a Highly Reactive Acysulfonamide "Safety Catch" Linker for Solid-Phase Synthesis" 118:3055-3056 (1996).|
|10||Bains, DNA sequencing by mass spectrometry: Outline of a potential future application, Chimicaoggi 9:13-16 (1991).|
|11||Bains, Setting a sequence to sequence a sequence, Biotechnology 10:757-758 (1992).|
|12||Barmwarth, Solid-phase synthesis of oligodeoxynucleotides containing phosphoramidate internucleotide linkages and their specific chemical cleavage, Helvctica Chimica Acta 71:1517-1527 (1988).|
|13||Barrell, DNA sequencing: present limitations and prospects for the future, FASEB J. 5:40-45 (1991).|
|14||Batista-Viera et al., A new method for reversible immobilization of thiol biomolecules based on solid-phase bound thiolsulfonate groups, App. Biochem and Biotech, 31:175-195 (1991).|
|15||Beaucage et al., The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications, Tetrahedron 49:6123-6194 (1993).|
|16||Beck and Koster, Applications of dioxetane chemiluminescent probes to molecular biology, Anal. Chem. 62:2258-2270 (1990).|
|17||Beck et al., Chemiluminescent detection of DNA: application of DNA sequencing and hybridization, Nucleic Acids Res. 17(13):5115-5123 (1989).|
|18||Bray, A.M. et al., "Direct Cleavage of Peptides from a Solid Support into Aqueous Buffer. Application in Simultaneous Multiple Peptide Synthesis", J. Org. Chem., 56:6659-6666 (1991).|
|19||Brennan et al., New methods to sequence DNA by mass spectrometry, SPIE, vol. 1206, New Technol. Cytom. Mol. Biol. pp. 60-77 (1990).|
|20||Broude et al., Enhanced DNA sequencing by hybridization, Proc. Natl. Acad. Sci. 91:3072-3076 (1994).|
|21||Brown et al., A single-bead decode strategy using electrospray ionization mass spectrometry and a new photolabile linker: 3-Amino-3-(2-nitrophenyl)propionic acid, Molec. Diversity 1:4-12 (1995).|
|22||Burgess, K. et al., "An Approach to Photolabile, Fluorescent Protecting Groups", J. Org. Chem., 62:5165-5168 (1997).|
|23||Certified English language translation of "Correspondence between R. Mandrand and Mr. Daniel dated; Mar. 14, May 3, and Dec. 5, 1977".|
|24||Certified English language translation of International PCT Publication WO 92/04465.|
|25||Chen and Seeburg, Supercoil sequencing: A fast and simple method for sequencing plasmid DNA, DNA 4(2):165-170 (1985).|
|26||Chrisey et al., Covalent attachment of synthetic DNA to self-assembled monlayer films, Nucl. Acids Res. 24:3031-3039 (1996).|
|27||Chrisey et al., Fabrication of patterened DNA surfaces, Nucl. Acids Res. 24:3040-3047 (1996).|
|28|| *||Christensen, L. et al. "Solid-phase synthesis of peptide nucleic acids" J. Pept. Res. (1995) vol. 3, pp. 175-183.|
|29||Church et al., "Multiplex DNA Sequencing", Science 240:185-188 (1988).|
|30||Communication of Notice of Opposition (R. 57(1) EPC), with Grounds of Opposition, dated Jun. 24, 2002, for European Patent EP 0 937 097 B1.|
|31||Correspondence between R. Mandrand and Mr. Daniel dated; Mar. 14, May 3, and Dec. 5, 1977.|
|32||Correspondence regarding volume of wells in 611F96 plates, Oct. 26, 2005.|
|33||Crain, "Mass spectrometric techniques in nucleic acid research", Mass Spear. Rev. 9:505-554 (1990).|
|34||Curtis et al., IEEE Transactions on Nanobioscience, 3(1):61-65 (2004).|
|35||Damha, et al., An Improved Procedure for Derivatization of Controlled-Pore Glass Beads for Solid-Phase Oligonucleotide Synthesis, Nucleic Acids Research, (1990), pp. 3813-3821, vol. 18, No. 13, Oxford University Press.|
|36||Damha, Masad J. et al.; An Improved Procedure for Derivatization of Controlled-Pore Glass Beads for Solid-Phase Oligonucleotide Synthesis, Nucleic Acids Research 18(13):3813-3821 (1990).|
|37||DeGrado, W.F. and Kaiser, E.T., "Polymer-Bound Oxime Esters as Supports for Solid-Phase Peptide Synthesis. Preparation of Protected Peptide Fragments", J. Org. Chem., 45:1295-1300 (1980).|
|38||Derwent Publications, WPI Acc. No. #90-108917/199015 citing European Patent No. EP0360677 published Mar. 28, 1990.|
|39||Derwent Publications, WPI Acc. No. #90-133198/199018, citing German Patent No. DE3930312 published Apr. 26, 1990.|
|40||Derwent Publications, WPI Acc. No. #90-302767/199040, citing Japanese Patent No. JP221 5399 published Aug. 28, 1990.|
|41||Derwent Publications, WPI Acc. No.#90-321790/199043, citing German Patent No. DE4011991 published Oct. 18, 1990.|
|42||Derwent Publications, WPI Ace, No. #85-224987/198942, citing International PCT Application No. WO 89/09406 published Oct. 05, 1989.|
|43||Derwent Publications, WPI Ace. No. #88-311964/198844, citing Japanese Patent No. JP 63230086 published Sep. 1988.|
|44||Dolitzky et al., "Synthesis, Characterization, and Use of Immobilized Polyacrolein Microspheres in Diagnostics: A Model Determination of alpha-Antitrypsin in Human Serum", Anal. Biochem., 220:257-267, 1994.|
|45||Drmanac, et al., Sequencing of megabase plus DNA by hybridization: Theory of the method, Genomics 4:114-128 (1989).|
|46||Eckstein and Goody, Synthesis and properties of diastereoisomers of adenosine 5'-(0-2-thiotriphosphate)and adenosine 5'-(0-2-thiotriposphate), Biochemistry 15(8):1685-1691 (1976).|
|47||Eckstein, F., Phosphorothioate analogues of nucleotides, Accounts Chem. Res. 12:204-210 (1979).|
|48||Eckstein, Nucleoside phosphorothioates, Ann. Rev. Biochem. 54:367-402 (1985).|
|49||English translation of the statement of the opponents grounds of Appeal.|
|50||Frank and Koster, DNA chain length and the influence of base composition on electrophoretic mobility of oligodeoxyribonucleotides in polyacrylamide-gels, Nucl. Acids Res. 6:2069-2087 (1979).|
|51||Fu et al., "A DNA sequencing strategy that requires only five bases of known terminalsequencing for priming", Proc. Natl. Acad. Sci. 92:10162-10166 (1995).|
|52||Fujita et al., Surprising lability of biotin-streptavidin bond during transcription ofbiotinylated DNA bound to paramagnetic beads,Bio Techniques 14:608-617 (1993).|
|53||Gait, M.J., ed., "Oliqonucleotide Synthesis : A Practical Approach", IRL Practical Approach Series, IRL Press, Oxford, 1984, Table of Contents only.|
|54||Gayo, L.M. and Suto, M.J., "Traceless Linker: Oxidative Activation and Displacement ofa Sulfur-Based Linker", Tetrahedroon Lett., 38(2):211-214 (1997).|
|55||Gehring et al., "Enzyme-linked immunomagnetic electrochemical detection of Salmonella typhimurium", J. Immun. Methods, 195:15-25, 1996.|
|56||Ghosh and Musso, Covalent attachment to solid supports, Nucl. Acids Res. 15:5353-5372 (1987).|
|57||Gildea et al., A versatile acid-labile linker for modification of synthetic biomolecules, Tetrahedron Letters 31:7095-7398 (1990).|
|58||Goldmacher et al., Photoactivation of toxin conjugates, Bioconjuqate Chem. 3:104-107, (1992).|
|59||Greene, Protective Groups in Organic Synthesis, 2nd Edition, Wiley & Sons, table of Contents (1991).|
|60||Han, Y. et al., "Silicon Directed ipso-Substitution of Polymer Bound Arylsilanes: Preparation of Biaryls via the Suzuki Cross-Coupling Reaction", Tetrahedron Lett., 37(16):2703-2706 (1996).|
|61||Hayashi et al., Immobilization of thiol proteases onto porous poly(vinyl alchol) beads, Polymer Journal, 25:5, 489-497 (1993).|
|62||Hayashi, et al., Immobilization of Thiol Proteases onto Porous Poly(vinyl Alchol) Beads, Polymer Journal, (1983), pp. 489-497, vol. 25, No. 5.|
|63||Hazum et al. A photocleavable protecting group for the function of cysteine, Pept. Proc. Eur. Pept. Symp. 16th Brunfeldt, K (Ed), pp. 105-110 (1981).|
|64||Hermanson, Bioconjugate Techniques, Academic Press (1996) TOC only.|
|65||Higuchi et al., A general method of in vitro preparation and mutagenesis of DNA fragments: Study of protein and DNA interactions, Nucleic Acids Res.16:7351-7367 (1988).|
|66||Higuchi et al., Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions, Bio Technology 11:1026-1030 (1993).|
|67||Hillenkamp and Ehring, Laser desorption mass spectrometry Part 1: Basic mechanisms and techniques, Mass Spectrometry in the Biological Sciences: A tutorial, pp. 165-179 (1992).|
|68||Hillenkamp et al., "Matrix Assisted UV_Laser Desorption/ionization: A New Approach to Mass Spectrometry of Large Biomolecules", Bio Mass Spectr., Burlingame and McCloskey (eds.), pp. 49-61, Elsevier Science Publishers B.V., Amsterdman (1989).|
|69||Hobbs and Eckstein, A general method for the synthesis of 2'-azido-2'deoxy-and 2'-amino-2'-deoxyribofuranoxyl purines, J. Org. Chem. 42:714-719 (1976).|
|70||Hornes and Korsnes, Magnetic DNA Hybridization of Oligonucleotide probes attached to super paramagnetic beads and their use in the isolation of Poly(A) mRNA from Eukaryotic cells, GATA7:145-150 (1990).|
|71||Hultman et al., Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support, Nucl. Acids Res. 17:4937-4946 (1989).|
|72||Huth-Fehre et al., Matrix-assisted laser desorption mass spectrometry of oligodeoxythmidulic acids, Rapid Communications in Mass Spectrometry6(3):209-213 (1992).|
|73||Hyman, A new method of sequencing DNA, Anal. Biochem. 174:423-436 (1988).|
|74||Innis et al., editors, PCR Protocols: A guide to methods and applications, Academic Press, San Diego (1990), TOC only.|
|75||Innis et at., DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA polymerase chain-reaction-amplified DNA, Proc. Natl. Acad. Sci. USA85:9436-9440 (1988).|
|76||Jacobson, et al., Applications of mass spectrometry to DNA sequencing, GATA 8:223-229 (1991).|
|77||Jett et al., "High-Speed DNA Sequencing: An Approach Based Upon fluorescence Detection of Single Molecules", J. Bio Strut & Dynam. 7(2):301-09 (1989).|
|78||Jurinke, C. et al., "Recovery of Nucleic Acids from Immobilized Biotin-Streptavidin Complexes using Ammonium Hydroxide and Applications in MALDI-TOF Mass Spectrometry", Anal. Chem., 69:904-910 (1997).|
|79||Kaldor, S.W. et al., "Use of Solid Supported Nucleophiles and Electrophiles for the Purification of Non-Peptide Small Molecule Libraries", Tetrahedron Lett.,37(40):7193-7196 (1996).|
|80|| *||Livnah, O. et al. "Three-dimensional structures of aviding and the avidin-biotin complex" PNAS (1993) vol. 90, pp. 5076-5080.|
|81||Locasoio and Gaitan, Proceeding microTAS Conference, Netherlands May 14-18, 2000. Conference Article.|
|82||Lund, et al., Assessment of Methods for Covalent Binding of Nucleic Acids to Magnetic Beads, Dynabeads(TM), and the Characteristics of the Bound Nucleic Acids In Hybridization Reactions, Nucleic Acids Research, (1988), pp. 10861-10880, vol. 16, No. 22.|
|83||Mansion, J.E., "Harrap's Shorter French and English Dictionary" 1967 pp. P:26 and W:11-12, Part One. French-English Eds., Ledesert and Ledesert, Pub. Bordas Paris, in association with George G. Harrap & Company, LTD.|
|84||Mansion, J.E., "Harrap's Shorter French and English Dictionary", 1967, pp. P:26 and W:11-W12, Part One, French-English, Ed. Ledesert, and Ledesert Pub. Bordas, Paris in association with George G. Harrap & Company LTD.|
|85||Margel et al., "Surface Modification. I. Polyacrolein Microspheres Covalently Bonded onto Polyethylene", J. Poly. Sci., 30:1103-1110, 1992.|
|86||Minutes and associated documents regarding European Patent Office Opposition Proceedings conducted Feb. 14, 2006.|
|87||Morrison and Boyd, Organic Chemistry, 5th ed., 1987, p. 1.|
|88||Opponent Letter, Nov. 2, 2005.|
|89||Opposition Division Communication Re: Opponent Opposition, Jun. 7, 2004.|
|90||Opposition Division Preliminary Opinion, Apr. 2, 2003.|
|91||Opposition Division Second Opinion, Mar. 18, 2004.|
|92||Opposition Proceedings New Main request, May 24, 2004.|
|93||Opposition Proceedings New Main request, Oct. 13, 2003.|
|94||Patentee's Observations on the Opposition filed with the European Patent Office on Jan. 3, 2003.|
|95||Pon, et al., Derivitization of Controlled Pore Glass Beads for Solid Phase Oligonucleotide Synthesis, BioTechniques, (1988), pp. 768-770, 773-775; vol. 6, No. 8.|
|96||Response to Oral Proccedings Summons, Nov. 7, 2005.|
|97||Ruppert et al., "Preparation of plasmid DNA as Sequencing Templates in a Microliter Plate Format" Paper presented, Cold Spring Harbor Laboratory, N.Y., USA, May 10-14, 1995.|
|98||Schriemer et al., "Combining Avidin-Biotin Chemistry with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry", Anal. Chem., 68:3382-3387, 1996.|
|99||Stedman's Medical Dictionary, 26th Edition, 1995, pp. 375-376.|
|100||Taber's Cyclopedic Medical Dictionary, 14th edition, 1983, p. 321.|
|101|| *||Wahlberg, J. et al "Automated magnetic preparation of DNA . . . " Electrophoresis (1992) vol. 13, pp. 547-551.|
| Patente citante|| Fecha de presentación|| Fecha de publicación|| Solicitante|| Título|
|USRE44693||22 Jul 2009||7 Ene 2014||Sequenom, Inc.||Beads bound to a solid support and to nucleic acids|
| || |
| Clasificación de EE.UU.||435/6.12, 536/25.4, 436/501, 435/288.4, 536/24.3, 536/25.3, 435/6.1|
| Clasificación internacional||C40B60/14, G01N35/10, C07H21/00, C40B40/06, C40B40/10, C07F9/24, C12Q1/68, C07H21/04|
| Clasificación cooperativa||B01J2219/00648, B01J2219/00387, B01J2219/00596, B01J2219/0072, B01J2219/00659, B01J2219/00511, B01J2219/00527, B01J2219/00468, C40B40/06, B01J2219/00725, B01J2219/005, B01J2219/00315, C12Q1/6872, C12Q1/6837, B01J2219/00497, B01J2219/0052, B01J2219/00317, G01N35/1067, B01J2219/00504, B01J2219/00585, C40B60/14, G01N2035/1069, C07H21/00, C40B40/10, B01J2219/00722|
| Clasificación europea||C07H21/00, C12Q1/68B10A, C12Q1/68E2|
|27 Jun 2014||AS||Assignment|
Owner name: AGENA BIOSCIENCE, INC., CALIFORNIA
Free format text: CHANGE OF NAME;ASSIGNOR:BIOSCIENCES ACQUISITION COMPANY;REEL/FRAME:033248/0073
Effective date: 20140530
|4 Abr 2012||FPAY||Fee payment|
Year of fee payment: 12