U.S. patent number RE41,005 [Application Number 10/277,021] was granted by the patent office on 2009-11-24 for beads bound to a solid support and to nucleic acids.
This patent grant is currently assigned to Sequenom, Inc.. Invention is credited to Hubert Koster, David M. Lough.
United States Patent |
RE41,005 |
Koster , et al. |
November 24, 2009 |
Beads bound to a solid support and to nucleic acids
Abstract
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.
Inventors: |
Koster; Hubert (Figino,
CH), Lough; David M. (Cambridge, MA) |
Assignee: |
Sequenom, Inc. (San Diego,
CA)
|
Family
ID: |
41327979 |
Appl.
No.: |
10/277,021 |
Filed: |
October 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
08746036 |
Nov 6, 1996 |
5900481 |
|
|
Reissue of: |
08933792 |
Sep 19, 1997 |
06133436 |
Oct 17, 2000 |
|
|
Current U.S.
Class: |
435/6.12;
435/288.4; 435/6.1; 436/501; 536/24.3; 536/25.3; 536/25.4 |
Current CPC
Class: |
C12Q
1/6872 (20130101); C12Q 1/6837 (20130101); C07H
21/00 (20130101); C12Q 1/6837 (20130101); C12Q
1/6837 (20130101); C12Q 1/6837 (20130101); C12Q
1/6872 (20130101); B01J 2219/00315 (20130101); B01J
2219/00317 (20130101); G01N 2035/1069 (20130101); G01N
35/1067 (20130101); C40B 60/14 (20130101); C40B
40/10 (20130101); C40B 40/06 (20130101); B01J
2219/00725 (20130101); B01J 2219/00722 (20130101); B01J
2219/0072 (20130101); B01J 2219/00659 (20130101); B01J
2219/00387 (20130101); B01J 2219/00468 (20130101); B01J
2219/00497 (20130101); B01J 2219/005 (20130101); B01J
2219/00504 (20130101); B01J 2219/00511 (20130101); B01J
2219/0052 (20130101); B01J 2219/00527 (20130101); B01J
2219/00585 (20130101); B01J 2219/00596 (20130101); B01J
2219/00648 (20130101); C12Q 2525/197 (20130101); C12Q
2523/10 (20130101); C12Q 2531/113 (20130101); C12Q
2565/537 (20130101); C12Q 2523/10 (20130101); C12Q
2565/627 (20130101); C12Q 2565/507 (20130101); C12Q
2565/518 (20130101); C12Q 2535/101 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C07H 21/04 (20060101) |
Field of
Search: |
;536/24.3,25.3,23.1,25.4
;525/332.2 ;502/233 ;435/6 ;436/94 |
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|
Primary Examiner: Maier; Leigh C.
Attorney, Agent or Firm: Grant Anderson LLP
Parent Case Text
RELATED APPLICATIONS
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.
Claims
We claim:
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 .mu.m 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: i) beads, ii) an insoluble support, and iii)
conjugation means for linking nucleic acids to the beads and the
beads to the support.Iadd., wherein the solid support is selected
from the group consisting of arrays of pits and multiwell supports
comprising nanoliter wells.Iaddend..
.[.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 .Iadd.and the solid support is
selected from the group consisting of arrays of pits and multiwell
supports comprising nanoliter wells.Iaddend..
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 a.]. .Iadd.The composition of
claim 1, wherein the .Iaddend.nucleic acid .[.molecule comprising
protein.]. .Iadd.comprises a peptide .Iaddend.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.Iadd., and the solid
support is selected from the group consisting of arrays of pits and
multiwell supports comprising nanoliter wells.Iaddend..
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..].
.Iadd.24. The composition of claim 1, wherein the solid support
comprises an array of pits..Iaddend.
.Iadd.25. The composition of claim 1, wherein beads are conjugated
to the support in pits on the array..Iaddend.
.Iadd.26. The composition of claim 1, wherein the solid support is
a multiwell support comprising nanoliter wells..Iaddend.
.Iadd.27. The composition of claim 1, wherein beads are conjugated
to the support in wells on the support..Iaddend.
.Iadd.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..Iaddend.
.Iadd.29. The composition of claim 28, wherein interaction is an
ionic interaction..Iaddend.
.Iadd.30. The composition of claim 28, wherein the interaction is a
covalent interaction..Iaddend.
.Iadd.31. The composition of claim 28, wherein the interaction is a
polar interaction..Iaddend.
.Iadd.32. The composition of claim 28, wherein the interaction is a
hydrophobic interaction..Iaddend.
.Iadd.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..Iaddend.
.Iadd.34. The method of claim 33, wherein the solid support
comprises an array of pits..Iaddend.
.Iadd.35. The method of claim 34, wherein beads are conjugated to
the support in pits on the array..Iaddend.
.Iadd.36. The method of claim 33, wherein the solid support is a
multiwell support comprising nanoliter wells..Iaddend.
.Iadd.37. The method of claim 36, wherein beads are conjugated to
the support in wells on the support..Iaddend.
.Iadd.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..Iaddend.
.Iadd.39. The method of claim 38, wherein the solid support
comprises an array of pits..Iaddend.
.Iadd.40. The method of claim 39, wherein beads are conjugated to
the support in pits on the array..Iaddend.
.Iadd.41. The method of claim 38, wherein the solid support is a
multiwell support comprising nanoliter wells..Iaddend.
.Iadd.42. The method of claim 41, wherein beads are conjugated to
the support in wells on the support..Iaddend.
.Iadd.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..Iaddend.
.Iadd.44. The composition of claim 1, wherein the nucleic acid is
single-stranded..Iaddend.
.Iadd.45. The process of claim 8, wherein the nucleic acid is
single-stranded..Iaddend.
.Iadd.46. The method of claim 33, wherein the nucleic acid is
single-stranded..Iaddend.
.Iadd.47. The method of claim 38, wherein the nucleic acid is
single-stranded..Iaddend.
.Iadd.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..Iaddend.
.Iadd.49. The method of claim 48, wherein the nucleic acid
conjugated to the beads is single-stranded..Iaddend.
.Iadd.50. The method of claim 48, wherein the solid support
comprises an array of pits..Iaddend.
.Iadd.51. The method of claim 50, wherein beads are conjugated to
the support in pits on the array..Iaddend.
.Iadd.52. The method of claim 48, wherein the solid support is a
multiwell support comprising nanoliter wells..Iaddend.
.Iadd.53. The method of claim 52, wherein beads are conjugated to
the support in wells on the support..Iaddend.
.Iadd.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..Iaddend.
.Iadd.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..Iaddend.
.Iadd.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..Iaddend.
.Iadd.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..Iaddend.
.Iadd.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..Iaddend.
.Iadd.59. The method of claim 48, wherein the nucleic acid is
detected by a spectrometric method..Iaddend.
.Iadd.60. The method of claim 59, wherein the spectrometric method
comprises fluorescence detection..Iaddend.
.Iadd.61. The method of claim 48, wherein the captured target
nucleic acid is from a biological sample..Iaddend.
.Iadd.62. The method of claim 48, wherein the nucleic acid
conjugated to the beads is DNA..Iaddend.
.Iadd.63. The method of claim 48, wherein the nucleic acid
conjugated to the beads is RNA..Iaddend.
.Iadd.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..Iaddend.
.Iadd.65. The method of claim 64, wherein the nucleic acid bound to
the beads is single-stranded..Iaddend.
.Iadd.66. The method of claim 64, wherein the nucleic acid bound to
the beads is DNA..Iaddend.
.Iadd.67. The method of claim 64, wherein the nucleic acid is
detected by a spectrometric method..Iaddend.
.Iadd.68. The method of claim 67, wherein the spectrometric method
comprises fluorescence detection..Iaddend.
.Iadd.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..Iaddend.
.Iadd.70. The method of claim 69, wherein the interaction is an
ionic interaction..Iaddend.
.Iadd.71. The method of claim 69, wherein the interaction is a
polar interaction..Iaddend.
.Iadd.72. The method of claim 69, wherein the interaction is a
hydrophobic interaction..Iaddend.
.Iadd.73. The method of claim 69, wherein the solid support is a
glass surface..Iaddend.
.Iadd.74. The method of claim 69, wherein the solid support is a
support with an array of pits..Iaddend.
.Iadd.75. The method of claim 69, wherein the solid support is a
support with nanoliter wells..Iaddend.
.Iadd.76. The method of claim 69, wherein the solid support is a
silicon wafer..Iaddend.
.Iadd.77. The method of claim 69, wherein the capture nucleic acid
is DNA..Iaddend.
.Iadd.78. The method of claim 69, wherein the capture nucleic acid
is RNA..Iaddend.
.Iadd.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..Iaddend.
.Iadd.80. The composition of claim 79, wherein the interaction is
an ionic interaction..Iaddend.
.Iadd.81. The composition of claim 79, wherein the interaction is a
polar interaction..Iaddend.
.Iadd.82. The composition of claim 79, wherein the interaction is a
hydrophobic interaction..Iaddend.
.Iadd.83. The composition of claim 79, wherein the solid support is
a glass surface..Iaddend.
.Iadd.84. The composition of claim 79, wherein the solid support is
a support with an array of pits..Iaddend.
.Iadd.85. The composition of claim 79, wherein the solid support is
a support with nanoliter wells..Iaddend.
.Iadd.86. The composition of claim 79, wherein the solid support is
a silicon wafer..Iaddend.
.Iadd.87. The composition of claim 79, wherein the nucleic acid
bound to the beads is DNA..Iaddend.
.Iadd.88. The composition of claim 19, wherein the nucleic acid
bound to the beads is RNA..Iaddend.
Description
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., Sephadex.sup.R), and
agarose (e.g., Sepharose.sup.R). In a preferred embodiment, the
bead is of a size in the range of about 1 to about 100 .mu.m 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 Dynabeads.sup.R, Wang resin;
Merrifield resin, which is chloromethylated
copolystyrene--divinylbenzene(DVB) resin,
Sephadex.sup.R/Sepharose.sup.R 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, Sephadex.sup.R/
Sepharose.sup.R, cellulose, magnetic beads, Dynabeads.sup.R, 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
TABLE-US-00001 TABLE 1 LINKER/INTERACTION EXAMPLES
streptavidin-biotin.sup.a, c/photolabile biotin.sup.b biotinylated
pin, avidin beads, photolabile biotin DNA hydrophobic.sup.a
C18-coated pin, tritylated DNA magnetic.sup.a electromagnetic pin,
steptavidin Dynabeads, biotin DNA acid-labile linker.sup.b glass
pin, bifunctional trityl- linked DNA amide bond(s).sup.c silicon
wafer, Wang resin, amino-linked DNA disulfide bond.sup.a silicon
wafer, beads are bound on the flat surface forming arrays or in
arrays of nanoliter wells, thiol beads, thiolated DNA
photocleavable bond/linker thioether bond.sup.c silicon wafer,
beads are bound on the flat surface forming arrays or in arrays of
nanoliter wells, thiolated DNA .sup.aThese interactions are
reversible .sup.bThese non-reversible interactions are rapidly
cleaved .sup.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.times.A, C, G, and
T). The sequencing reactions can be carried out in a volume of 10
.mu.l 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.times.5 cm in a 12.times.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.times.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 4-800.mu. across
.times.100.mu. 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 .mu.m 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.degree. 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
.mu.l of 3-HPA matrix at room temperature for 2 min. An aliquot of
0.5 .mu.l 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 .mu.l of 50 mM ammonium citrate was added to
the beads. After a 5 min. incubation at room temperature, 1 .mu.l
of 3-HPA matrix was added and 0.5 .mu.l 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 .mu.l of 3-HPA matrix and 0.5 .mu.l 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
Equivalents
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.
* * * * *
References