U.S. patent application number 10/210307 was filed with the patent office on 2003-07-31 for methods of immobilizing ligands on solid supports and apparatus and methods of use therefor.
This patent application is currently assigned to Matrix Technologies Corporation. Invention is credited to Abrams, Ezra S., Mielewczyk, Slawomir, Patterson, Brian C., Zhang, Tianhong.
Application Number | 20030143569 10/210307 |
Document ID | / |
Family ID | 26848471 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143569 |
Kind Code |
A1 |
Abrams, Ezra S. ; et
al. |
July 31, 2003 |
Methods of immobilizing ligands on solid supports and apparatus and
methods of use therefor
Abstract
A method is provided for immobilizing a ligand, e.g., a nucleic
acid, on a solid support. The method includes providing a solid
support containing an immobilized latent thiol group, activating
the thiol group, contacting the activated thiol group with a
nucleic acid comprising an acrylamide functional group, and forming
a covalent bond between the two groups, thereby immobilizing the
nucleic acid to the solid support. Kits containing the solid
supports and method of utilizing the solid supports are also
provided.
Inventors: |
Abrams, Ezra S.; (W. Newton,
MA) ; Zhang, Tianhong; (Framingham, MA) ;
Mielewczyk, Slawomir; (Newton, MA) ; Patterson, Brian
C.; (Waltham, MA) |
Correspondence
Address: |
WOOD, HERRON & EVANS, L.L.P.
2700 Carew Tower
441 Vine St.
Cincinnati
OH
45202
US
|
Assignee: |
Matrix Technologies
Corporation
Hudson
NH
|
Family ID: |
26848471 |
Appl. No.: |
10/210307 |
Filed: |
August 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10210307 |
Aug 1, 2002 |
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09649637 |
Aug 28, 2000 |
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6492118 |
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60151267 |
Aug 27, 1999 |
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60177844 |
Jan 25, 2000 |
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Current U.S.
Class: |
435/6.11 ;
427/2.11; 435/6.12 |
Current CPC
Class: |
G01N 33/54353 20130101;
C12Q 1/6834 20130101; C12Q 1/6837 20130101; C07H 21/04 20130101;
C12Q 1/6834 20130101; C12Q 2523/107 20130101; C12Q 1/6837 20130101;
C12Q 2523/107 20130101 |
Class at
Publication: |
435/6 ;
427/2.11 |
International
Class: |
C12Q 001/68; B05D
003/00 |
Claims
What is claimed is:
1. A method of immobilizing an affinity ligand on a solid support
comprising: providing a solid support comprising an immobilized
thiol group; contacting the thiol group with a nucleic acid
comprising an acrylamide functional group; and forming a covalent
bond between the two groups, thereby immobilizing the ligand on the
solid support.
2. The method of claim 1, wherein the ligand is selected from the
group consisting of a nucleic acid, a modified nucleic acid and a
nucleic acid analog.
3. The method of claim 2, wherein the solid support comprises a
plurality of thiol groups
4. The method of claim 3, wherein a plurality of ligands are
immobilized on the solid support.
5. The method of claim 4, wherein the solid support is formed from
a compound selected from the group consisting of class, plastic and
metal.
6. The method of claim 5, wherein the solid support comprises two
or more spatially distinct regions, each region comprising a
plurality of immobilized nucleic acids.
7. The method of claim 6, wherein the solid support further
comprises a polymer layer.
8. The method of claim 7, wherein the solid support comprises a
microarray.
9. The method of claim 1, wherein the thiol groups comprise reduced
disulfide groups.
10. A method of immobilizing an affinity ligand on a solid support
comprising the steps of: providing a solid support comprising
immobilized latent thiol groups; activating the latent thiol
groups; and reacting the activated thiol groups with an affinity
ligand having at least one acrylamide functional group, thereby
immobilizing an affinity ligand on a solid support.
11. The method of claim 10, wherein the ligand is selected from the
group consisting of a nucleic acid, a modified nucleic acid and a
nucleic acid analog.
12. The method of claim 11, wherein the steps of activating the
latent thiol groups and reacting the activated thiol groups occur
essentially simultaneously.
13. The method of claim 12, wherein the solid support is formed
from a compound selected from the group consisting of glass,
plastic and metal.
14. The method of claim 13, wherein the solid support comprises two
or more spatially distinct regions, each region comprising a
plurality of immobilized nucleic acids.
15. The method of claim 14, wherein the solid support further
comprises a polymer layer.
16. The method of claim 15, wherein the solid support comprises a
microarray.
17. The product formed by the method of claim 10.
18. A method of immobilizing an affinity ligand on microarray
comprising the steps of: providing a solid support comprising
immobilized latent thiol groups; activating the latent thiol
groups; and reacting the activated thiol groups with an affinity
ligand having at least one .alpha.,.beta. unsaturated carbonyl
functional group, thereby immobilizing an affinity ligand on a
solid support.
19. The method of claim 18, wherein the ligand is selected from the
group consisting of a nucleic acid, a modified nucleic acid and a
nucleic acid analog.
20. The method of claim 19, wherein the steps of activating the
latent thiol groups and reacting the activated thiol groups occur
essentially simultaneously.
21. The method of claim 10, further comprising the steps of:
contacting a glass solid support with a silane compound represented
by the following structural formula: 27wherein: X is a halogen; and
RI, R, and R, are each, independently, a halogen, an alkyl group.
an alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, provided that at least one of
RI, R, or R, is an alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, thereby forming a solid
support having an unsaturated aliphatic surface; and contacting the
unsaturated aliphatic surface of the solid support with a
polymerization solution containing free radical initiator,
disulfide bisacrylamide, and optionally an acrylamide, wherein the
disulfide bisacrylamide is represented by the following structural
formula: 28wherein n and m are each, independently, a positive
integer, thereby forming a solid support comprising immobilized
latent thiol groups.
22. The method of claim 21, wherein the latent thiol groups are
activated by contacting the solid support with a disulfide reducing
agent.
23. The method of claim 21, wherein the polymerization solution
further includes alkylene bisacrylamide.
24. The method of claim 21, wherein the free radical initiator is
added to the polymerization solution after the solution is in
contact with the unsaturated aliphatic surface of the solid
support.
25. The method of claim 21, further comprising the step of
derivatizing a solid support with a latent thiol group, thereby
forming a solid support having immobilized latent thiol groups.
26. The method of claim 21, wherein the solid support has an amine
functional group and the solid support is derivatized by contacting
the solid support with a compound represented by the following
structural formula: 29wherein: Y is a leaving group; L is a linking
group; and R.sub.4 is a thiol protecting group, thereby forming a
solid support having immobilized latent thiol groups.
27. The method of claim 26 wherein Y consists of a group selected
from the following groups: 30wherein R.sub.6 and R.sub.7 are
aliphatic groups.
28. The method of claim 26, wherein R, is selected from the group
consisting of: 31wherein R.sub.8 is a substituted or unsubstituted
aliphatic group, a substituted or unsubstituted aromatic group, a
substituted or unsubstituted heteroaromatic group, a substituted or
unsubstituted aralkyl, or a substituted or unsubstituted
heteroaralkyl group.
29. A method of preparing a solid support having immobilized thiol
groups, comprising the steps of: contacting a glass solid support
with a silane compound represented by the following structural
formula: 32wherein: X is a halogen; and R.sub.1, R.sub.2, and
R.sub.3, are each, independently, a halogen, an alkyl group, an
alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, provided that at least one of
R.sub.1, R.sub.2, or R.sub.3 is an alkenyl group or a group having
at least one .alpha.,.beta.-unsaturated carbonyl, thereby forming a
solid support having an unsaturated aliphatic surface; contacting
the unsaturated aliphatic surface of the solid support with a
polymerization solution containing free radical initiator,
disulfide bisacrylamide, and optionally an acrylamide, wherein the
disulfide bisacrylamide is represented by the following structural
formula: 33wherein: n and m are each, independently, a positive
integer, thereby forming a solid support comprising immobilized
latent thiol groups; and contacting the latent thiol groups with a
disulfide reducing agent, thereby forming a solid support having
immobilized thiol groups.
30. The method of claim 29, wherein a plurality of nucleic acids
are immobilized on the solid support.
31. The method of claim 30, wherein the solid support comprises two
or more spatially distinct regions, each region comprising a
plurality of immobilized nucleic acids.
32. The method of claim 29, wherein the thiol groups comprise
disulfide groups.
33. The method of claim 31, wherein the latent thiol groups in
selected regions of the support are activated, thereby providing a
support comprising selected regions of reactive thiol groups.
34. A method of forming an array of nucleic acids immobilized on a
solid support comprising: forming an amine-derivatized region on
the support; treating the amine-derivatized region with a
thiolating agent such that latent thiol groups immobilized on the
support are formed; activating the latent thiol groups; contacting
the activated thiol groups with a plurality of nucleic acids
comprising acrylamide functional groups; and forming a covalent
bond between the two groups, thereby forming an array of nucleic
acids immobilized on the solid support.
35. The method of claim 34, wherein each nucleic acid comprises a
nucleotide sequence substantially identical to the nucleotide
sequence of the other nucleic acids of the array.
36. The method of claim 34, wherein nucleic acids with a plurality
of nucleotide sequences are contained in the array.
37. The method of claim 34 comprising a plurality of
amine-derivatized regions.
38. The method of claim 34 further comprising a step of blocking
unbonded reactive thiol groups remaining following the binding of
the nucleic acids to the thiol groups.
39. The microarray prepared by the method of claim 34.
40. A kit for attaching nucleic acids to a solid support comprising
a solid support component comprising a plurality of immobilized
latent thiol groups and instructions for activating the thiol
groups to form covalent bonds with nucleic acids comprising
acrylamide functional groups.
41. The kit of claim 40 further comprising at least one component
selected from the group consisting of an activator component, an
acrylamide functional nucleic acids component, a blocking
component, a wash buffer and a wash buffer.
42. A kit for attaching nucleic acids to a solid support comprising
a solid support component comprising a plurality of immobilized
thiol groups and nucleic acids comprising acrylamide functional
groups.
43. The kit of claim 42, wherein the nucleic acids are immobilized
on a solid support by a covalent bond between the immobilized thiol
groups and the nucleic acids.
44. The kit of claim 43 further comprising at least one component
selected from the group consisting of an activator component, an
acrylamide functional nucleic acids component, a blocking component
and a wash buffer.
45. A method for detecting or separating target nucleic acids from
other components contained in a sample comprising: providing a
solid support comprising a plurality of immobilized nucleic acids
comprising nucleotide sequences complementary to a subsequence of
the nucleotide sequence of the target nucleic acid, wherein the
nucleic acids are immobilized by a covalent bond formed between a
thiol group immobilized on the solid support and an acrylamide
functional group contained on the nucleic acid; contacting the
immobilized nucleic acid with the sample; and hybridizing target
nucleic acids to immobilized nucleic acids with complementary
subsequences, thereby detecting or separating target nucleic acids
from other components contained in the sample.
46. The method of claim 45, wherein the target nucleic acids are
amplified after detection or separation.
47. The method of claim 45, wherein the method is used in an assay
selected from the group of assays for detecting a contaminant in a
sample, for medical diagnosis of a medical condition, for genetic
and physical mapping of genomes, for monitoring gene expression and
for DNA sequencing.
48. A method for detecting or separating target nucleic acids from
other components contained in a sample comprising: providing a
solid support comprising a plurality of immobilized thiol groups;
contacting the thiol groups with a plurality of nucleic acids
comprising nucleotide sequences complementary to a subsequence of
the nucleotide sequence of the target nucleic acid and acrylamide
functional groups; forming a covalent bond between the thiol and
acrylamide functional groups, thereby immobilizing the nucleic
acids on the solid support; contacting the immobilized nucleic
acids with the sample; and hybridizing target nucleic acids to
immobilized nucleic acids with complementary subsequences, thereby
detecting or separating target nucleic acids from other components
contained in the sample.
49. The method of claim 48, wherein the target nucleic acids are
amplified after detection or separation.
50. The method of claim 49, wherein the method is used in an assay
selected from the group of assays for detecting a contaminant in a
sample, for medical diagnosis of a medical condition, for genetic
and physical mapping of genomes, for monitoring gene expression and
for DNA sequencing.
51. The method of claim 10, wherein the solid support is doped or
undoped silica, alumina, quartz or glass, and wherein the method
further comprises the steps of: contacting the solid support with a
compound comprising a silane group or a carboxylic acid and a
substituted or unsubstituted alkenyl group or a group having at
least one (.alpha.,.beta.-unsaturated carbonyl, thereby forming a
solid support having an unsaturated aliphatic surface; and
contacting the unsaturated aliphatic surface of the solid support
with a polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing an acrylamide,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 34wherein: n and m are each, independently, a
positive integer, thereby forming a solid support comprising
immobilized latent thiol groups.
52. The method of claim 51, wherein the compound is represented by
the following structural formula: 35wherein: X is a halogen; and
RI, R2 and R3 are each, independently, a halogen, a substituted or
unsubstituted alkyl group, a substituted or unsubstituted alkenyl
group or a group having at least one a,P-unsaturated carbonyl,
provided that at least one of RI, R2 or R3 is a substituted or
unsubstituted alkenyl group or a group having at least one
a,p-unsaturated carbonyl.
53. The method of claim 51, wherein the latent thiol groups are
activated by contacting the solid support with a disulfide reducing
agent.
54. The method of claim 51, wherein the polymerization solution
further includes alkylene bisacrylamide.
55. The method of claim 51, wherein the free radical initiator is
added to the polymerization solution after the solution is in
contact with the unsaturated aliphatic surface of the solid
support.
56. The method of claim 10, wherein the solid support is selected
from the group consisting of gold, silver, copper, cadmium, zinc,
palladium, platinum, mercury, lead, iron, chromium, manganese,
tungsten, and alloys thereof, and wherein the method further
comprises the steps of: contacting the solid support with a
compound comprising a thiol group, sulfide or disulfide group and a
substituted or unsubstituted alkenyl group or a group having at
least one (x,p-unsaturated carbonyl, thereby forming a solid
support having an unsaturated aliphatic surface; and contacting the
unsaturated aliphatic surface of the solid support with a
polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing a conmonomer,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 36wherein: n and m are each, independently, a
positive integer, thereby forming a solid support comprising
immobilized latent thiol groups.
57. The method of claim 10, wherein the solid support is selected
from the group consisting of platinum or palladium, and wherein the
method further comprises the steps of: contacting the solid support
with a compound comprising a nitrile or isonitrile group and a
substituted or unsubstituted alkenyl group or a group having at
least one a,p-unsaturated carbonyl, thereby forming a solid support
having an unsaturated aliphatic surface; and contacting the
unsaturated aliphatic surface of the solid support with a
polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing an acrylamide,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 37wherein: n and m in are each, independently,
a positive integer, thereby forming a solid support comprising
immobilized latent thiol groups.
58. The method of claim 10, wherein the solid support is copper,
and wherein the method further comprises the steps of: contacting
the solid support with a compound comprising a hydroxamic acid
group and a substituted or unsubstituted alkenyl group or a group
having at least one .alpha.,.beta.-unsaturated carbonyl, thereby
forming a solid support having an unsaturated aliphatic surface;
and contacting the unsaturated aliphatic surface of the solid
support with a polymerization solution containing free radical
initiator and disulfide bisacrylamide and optionally containing an
acrylamide, wherein the disulfide bisacrylamide is represented by
the following structural formula: 38wherein n and mn are each,
independently, a positive integer, thereby forming a solid support
comprising immobilized latent thiol groups.
59. The method of claim 10, wherein the solid support is a polymer
comprising a reactive functional group, and wherein the method
further comprises the steps of: contacting the solid support with a
compound comprising a functional group which can react to form a
bond with the reactive functional group and a substituted or
unsubstituted alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, thereby forming a solid
support having immobilized unsaturated aliphatic groups; and
contacting the unsaturated aliphatic groups of the solid support
with a polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing an acrylamide,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 39wherein: n and m are each, independently, a
positive integer, thereby forming a solid support comprising
immobilized latent thiol groups.
60. The method of claim 59, wherein the polymeric solid support is
polystyrene.
61. The method of claim 59, wherein hod of claim 59, wherein the
reactive functional group of the polymeric solid support is an
amine group or a hydroxyl group and the compound is represented by
the following structural formula: 40wherein: Y is a leaving group;
L is a linking group; and R.sub.10 is a substituted or
unsubstituted alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl.
62. The compound of claim 61, wherein Y is selected from the group
consisting of: 41wherein R.sub.6 and R.sub.7 are each,
independently, a substituted or unsubstituted alkyl group, a
substituted or unsubstituted alkenyl group, a substituted or
unsubstituted aromatic group, a substituted or unsubstituted
heteroaromatic group, a substituted or unsubstituted aralkyl, or a
substituted or unsubstituted heteroaralkyl group.
63. The method of claim 10, further comprising the step of
derivatizing a solid support with a latent thiol group, thereby
forming a solid support having immobilized latent thiol groups.
64. The method of claim 63, wherein the solid support is selected
from the group consisting of doped or undoped silica, alumina,
quartz or glass, and the solid support is derivatized by contacting
it with a compound comprising a silane group or a carboxylic acid
group and a latent thiol group.
65. The method of claim 63, wherein the solid support is selected
from the group consisting of platinum or palladium, and the solid
support is derivatized by contacting it with a compound comprising
a nitrile or isonitrile group and a latent thiol group.
66. The method of claim 63, wherein the solid support is a polymer
comprising reactive functional groups, and the solid support is
derivatized by contacting it with a compound comprising a
functional group which can react to form a bond with the reactive
functional group and a latent thiol group.
67. The method of claim 63, wherein the polymeric solid support is
polystyrene.
68. The method of claim 66, wherein the reactive functional group
of the polymeric solid support is an amine or a hydroxyl group and
the solid support is derivatized by contacting the solid support
with a compound represented by the following structural formula:
42wherein: Y is a leaving group; L is a linking group; and R.sub.4
is a thiol protecting group, thereby forming a solid support having
immobilized latent thiol groups.
69. The method of claim 68, wherein Y is selected form the group
consisting of: 43wherein R.sub.6 and R.sub.7 are each,
independently, a substituted or unsubstituted alkyl group, a
substituted or unsubstituted alkenyl group, a substituted or
unsubstituted aromatic group, a substituted or unsubstituted
heteroaromatic group, a substituted or unsubstituted aralkyl, or a
substituted or unsubstituted heteroaralkyl group.
70. The method of claim 68, wherein is selected from the group
consisting of: 44wherein R.sub.6is a substituted or unsubstituted
alkyl group, a substituted or unsubstituted alkenyl group, a
substituted or unsubstituted aromatic group, a substituted or
unsubstituted heteroaromatic group, a substituted or unsubstituted
aralkyl, or a substituted or unsubstituted heteroaralkyl group.
71. A method of making a solid support having immobilized thiol
groups, comprising the steps of: contacting a glass solid support
with a silane compound represented by the following structural
formula: 45wherein: X is a halogen; and R.sub.1, R.sub.2 and
R.sub.3 are each, independently, a halogen, an alkyl group, an
alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, provided that at least one of
RI, R, or R, is an alkenyl group or group having at least one
.alpha.,.beta.-unsaturated carbonyl, thereby forming a solid
support having an unsaturated aliphatic surface; contacting the
unsaturated aliphatic surface of the solid support with a
polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing an acrylamide,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 46wherein: n and m are each, independently, a
positive integer, thereby forming a solid support comprising
immobilized latent thiol groups; and contacting the latent thiol
groups with a disulfide reducing agent, thereby forming a solid
support having immobilized thiol groups.
Description
RELATED APPLICATION(S)
[0001] This application claims the benefit of the Provisional
Application with Serial No. 60/151,267 filed Aug. 27, 1999 and the
Provisional Application with Serial No. 60/177,844 filed Jan. 25,
2000. The teachings of both cited applications are incorporated
herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] A variety of assay systems utilize ligands, e.g., nucleic
acids, immobilized on the surface of a solid support. Effective
immobilization of the nucleic acids is difficult, both because a
range of materials are used to form the solid supports utilized in
these assays, and because individual assays have special
requirements. Therefore, although a number of attachment mechanisms
have been developed, none are universally acceptable and most
exhibit notable deficiencies. Among other drawbacks, present
methods tend to require large amounts of nucleic acids, have high
background noise levels or lack versatility (Duran et al. U.S. Pat.
No. 5,858,653 issued Jan. 12, 1999).
[0003] The reproducible production of solid supports containing
immobilized nucleic acids can also be problematic. For example, a
convenient method of attachment utilizes nucleic acids with
acrylamide functional groups which can be copolymerized to
polyacrylamide gel matrices by free radical polymerization.
However, oxidation can affect the copolymerization process
resulting in variability in the results achieved using different
supports, even when prepared using the same materials. Moreover,
long-term stability of supports containing immobilized ligands has
been difficult to achieve, often limiting the period of use to
shortly after preparation.
SUMMARY OF THE INVENTION
[0004] The present invention is based, at least in part, on the
discovery of a novel and convenient method of mobilizing a ligand,
e.g., a nucleic acid, on a solid support. The method utilizes a
covalent bond formed between a thiol group immobilized on the solid
support and an acrylamide functional group contained on the nucleic
acid to immobilize the nucleic acid to the support. In a particular
embodiment, the covalent bond formed is a sulfide, a thioether,
bond. The solid support can contain a polymer layer.
[0005] The method and the supports it produces are advantageous in
several respects. The method utilizes reagents which are both
readily available and compatible with the types of analysis
conducted with solid supports. Because the materials can be used in
aqueous solutions, the need for special skills and sophisticated
chemical apparatus are minimized. In addition, because the
materials and the supports they form are quite stable, the
reproducibility from support to support which has previously proved
so difficult to achieve can be realized. This stability also
permits the components forming the bond to be combined at different
times. For example, because solid supports containing the latent
thiol groups of the invention are extremely stable, they can be
produced under consistent conditions for use at a later time. Prior
to analysis, the latent thiol groups can be activated and contacted
with the acrylamide modified nucleic acids to form a support
containing immobilized nucleic acids. In a particular embodiment,
the thiol groups are activated by contact with a reducing
agent.
[0006] In one embodiment, the invention is directed to a method of
immobilizing an affinity ligand on a solid support comprising
providing a solid support comprising an immobilized thiol group,
contacting the thiol group with a nucleic acid comprising an
acrylamide functional group, and forming a covalent bond between
the two groups, thereby immobilizing the ligand on the solid
support.
[0007] In a particular embodiment; the ligand is a nucleic acid, a
modified nucleic acid or a nucleic acid analog. The solid support
can comprise a plurality of thiol groups. A plurality of ligands
can be immobilized on the solid support. In alternate embodiments,
the solid support is formed from glass, silica, ceramic, plastic or
metal compounds. The solid support can comprises two or more
spatially distinct regions, each region comprising a plurality of
immobilized nucleic acids. The solid support can further comprise a
polymer layer. In a particular embodiment, the solid support can
comprise a microarray. The thiol groups can comprise disulfide
groups.
[0008] In another embodiment, the invention is directed to a method
of immobilizing an affinity ligand on a solid support comprising
the steps of providing a solid support comprising immobilized
latent thiol groups, activating the latent thiol groups, and
reacting the activated thiol groups with an affinity ligand having
at least one acrylamide functional group, thereby immobilizing an
affinity ligand on a solid support.
[0009] In a particular embodiment, the ligand is selected from the
group consisting of a nucleic acid, a modified nucleic acid and a
nucleic acid analog. The steps of activating the latent thiol
groups and reacting the activated thiol groups can occur
essentially simultaneously. In alternate embodiments, the solid
support is formed from glass, ceramic, plastic and metal. The solid
support can comprise two or more spatially distinct regions, each
region comprising a plurality of immobilized nucleic acids. The
solid support can further comprises a polymer layer. The solid
support can comprise a microarray.
[0010] In another aspect, the invention is directed to the product
formed by the method of forming a solid support described
above.
[0011] In another embodiment, the invention is directed to a method
of immobilizing an affinity ligand on microarray comprising the
steps of providing a solid support comprising immobilized latent
thiol groups, activating the latent thiol groups, and reacting the
activated thiol groups with an affinity ligand having at least one
.alpha.,.beta. unsaturated carbonyl functional group, thereby
immobilizing an affinity ligand on a solid support. In a particular
embodiment, the ligand is selected from the group consisting of a
nucleic acid, a modified nucleic acid and a nucleic acid analog.
The steps of activating the latent thiol groups and reacting the
activated thiol groups canoccur essentially simultaneously.
[0012] In another embodiment, the invention is directed to a method
of immobilizing an affinity ligand on microarray comprising the
steps of providing a solid support comprising immobilized latent
thiol groups, the latent thiol groups and reacting the activated
thiol groups with an affinity ligand having at least one
.alpha.,.beta. unsaturated carbonyl functional group, thereby
immobilizing an affinity ligand on a solid support. In a particular
embodiment, the ligand is a nucleic acid, a modified nucleic acid
or a nucleic acid analog. The steps of activating the latent thiol
groups and reacting the activated thiol groups can occur
essentially simultaneously.
[0013] The method can additionally include contacting a glass solid
support with a silane compound to form a solid support having an
unsaturated aliphatic surface. The silane compound can be
represented by Structural Formula I: 1
[0014] In Structural Formula I, X is a halogen, and R.sub.1,
R.sub.2 and R.sub.3 are each, independently, a halogen, an alkyl
group, an alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, provided that at least one of
R.sub.1, R.sub.2 or R.sub.3 is an alkenyl group or a group having
at least one .alpha.,.beta.-unsaturated carbonyl. The unsaturated
aliphatic surface is then contacted with a polymerization solution
containing free radical initiator, a disulfide bisacrylamide, and
optionally containing an acrylamide to form a solid support
comprising immobilized latent thiol groups. Disulfide
bisacrylamides can be represented by Structural Formula IIA: 2
[0015] In Structural Formula IIA, n and m are each, independently,
a positive integer.
[0016] The latent thiol groups can be activated by contacting the
solid support with a disulfide reducing agent. When it is desirable
to have a crosslinked gel having immobilized thiol groups, the
polymerization solution can additionally include alkylene
bisacrylatide.
[0017] In an alternative embodiment, the unsaturated aliphatic
surface is then contacted with a polymerization solution containing
free radical initiator, a compound having a
.alpha.,.beta.-unsaturated carbonyl and a protected thiol group,
and optionally containing an acrylamide to form a solid support
comprising immobilized latent thiol groups. The compound having an
.alpha.,.beta.-unsaturated carbonyl and a protected thiol group
preferably can be represented by Structural Formulas IIB-IID: 3
[0018] In Structural Formulas IIB-IID, R.sub.11 and R.sub.4 are
defined as above. R.sub.14 is --(CH.sub.2).sub.p- or
--(OCH.sub.2CH.sub.2).sub.p-. In a preferred embodiment, R.sub.4 is
--SR.sub.15, wherein R.sub.15 is a substituted or unsubstituted
alkyl group, a substituted or unsubstituted aromatic group or a
substituted or unsubstituted aralkyl group.
[0019] In several embodiments of the invention, it is useful to
provide latent thiol groups through the use of polymerizable
disulfide compounds. As indicated in Structures IIB-D, such
compounds can be monofunctional or bifunctional with regard to the
.alpha.,.beta. unsaturated carbonyl group. A commercially available
example of a bifunctional disulfide reagent is BAC. An example of a
monofunctional disulfide reagent is AEMA (Schnaar, R. L. et al.,
1985, Analytical Biochemistry, 151:268-281). Additional
monofunctional acrylamide disulfide derivatives can be generated by
reacting BAC with the reducting agents .beta.-mercaptoethanol and
thioacetic acid, as shown if FIGS. 8 and 9.
[0020] In a particular embodiment, the free radical initiator is
added to the polymerization solution after the solution is in
contact with the unsaturated aliphatic surface of the solid
support.
[0021] The method can additionally include derivatizing the solid
support with a latent thiol group, thereby forming a solid support
having immobilized latent thiol groups. In a particular embodiment,
the solid support includes an amine functional group and the solid
support is derivatized by contacting the solid support with a
compound represented by Structural Formula III: 4
[0022] In Structural Formula III, Y is a leaving group, L is a
linking group, and R.sub.4 is a thiol protecting group. The
derivatized solid support formed has immobilized latent thiol
groups.
[0023] In a particular embodiment, Y is one of the following: 5
[0024] wherein R.sub.6 and R.sub.7 are each, independently, a
substituted or unsubstituted alkyl group, a substituted or
unsubstituted alkenyl group, a substituted or unsubstituted
aromatic group, a substituted or unsubstituted heteroaromatic
group, a substituted or unsubstituted aralkyl, or a substituted or
unsubstituted heteroaralkyl group.
[0025] In a particular embodiment, R.sub.4 is one of the following
groups: 6
[0026] wherein R.sub.8 is a substituted or unsubstituted alkyl
group, a substituted or unsubstituted alkenyl group, a substituted
or unsubstituted aromatic group, a substituted or unsubstituted
heteroaromatic group, a substituted or unsubstituted aralkyl, or a
substituted or unsubstituted heteroaralkyl group.
[0027] In another aspect, the invention is directed to a method of
preparing a solid support having immobilized thiol groups. The
method includes contacting a glass solid support with a silane
compound represented by Structural Formula I to form a solid
support having an unsaturated aliphatic surface. The unsaturated
aliphatic surface of the solid support is then contacted with a
polymerization solution containing free radical initiator, a
disulfide bisacrylamide represented by Structural Formula IIA-D,
and optionally containing an acrylamide to form a solid support
comprising immobilized latent thiol groups. The latent thiol groups
of the solid support are then contacted with a disulfide reducing
agent to form a solid support having immobilized thiol groups.
[0028] In one embodiment, the solid support is doped or undoped
silica, alumina, quartz or glass, and the method further comprises
the steps of contacting the solid support with a compound
comprising a silane group or a carboxylic acid and a substituted or
unsubstituted alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, thereby forming a solid
support having an unsaturated aliphatic surface, and contacting the
unsaturated aliphatic surface of the solid support with a
polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing an acrylamide,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 7
[0029] wherein n and m are each, independently, a positive integer,
thereby forming a solid support comprising immobilized latent thiol
groups.
[0030] The compound can be represented by the following structural
formula: 8
[0031] wherein X is a halogen, and R.sub.1, R.sub.2 and R.sub.3 are
each, independently, a halogen, a substituted or unsubstituted
alkyl group, a substituted or unsubstituted alkenyl group or a
group having at least one .alpha.,.beta.-unsaturated carbonyl,
provided that at least one of R.sub.1, R.sub.2 or R.sub.3 is a
substituted or unsubstituted alkenyl group or a group having at
least one .alpha.,.beta.-unsaturated carbonyl.
[0032] The latent thiol groups can be activated by contacting the
solid support with a disulfide reducing agent. The polymerization
solution can further include alkylene bisacrylamide. The free
radical initiator can be added to the polymerization solution after
the solution is in contact with the unsaturated aliphatic surface
of the solid support
[0033] The solid support can be gold, silver, copper, cadmium,
zinc, palladium, platinum, mercury, lead, iron, chromium,
manganese, tungsten, and alloys thereof, and the method can further
comprises the steps of contacting the solid support with a compound
comprising a thiol group, sulfide or disulfide group and a
substituted or unsubstituted alkenyl group or a group having at
least one .alpha.,.beta.-unsaturated carbonyl, thereby forming a
solid support having an unsaturated aliphatic surface, and
contacting the unsaturated aliphatic surface of the solid support
with a polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing a conmonomer,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 9
[0034] whereinn and m are each, independently, a positive integer,
thereby forming a solid support comprising immobilized latent thiol
groups.
[0035] The solid support can be platinum or palladium, and the
method can further comprises the steps of contacting the solid
support with a compound comprising a nitrile or isonitrile group
and a substituted or unsubstituted alkenyl group or a group having
at least one .alpha.,.beta.-unsaturated carbonyl, thereby forming a
solid support having an unsaturated aliphatic surface, and
contacting the unsaturated aliphatic surface of the solid support
with a polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing an acrylamide,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 10
[0036] wherein n and m are each, independently, a positive integer,
thereby forming a solid support comprising immobilized latent thiol
groups.
[0037] The solid support can be copper, and the method can further
comprise the steps of contacting the solid support with a compound
comprising a hydroxamic acid group and a substituted or
unsubstituted alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, thereby forming a solid
support having an unsaturated aliphatic surface, and contacting the
unsaturated aliphatic surface of the solid support with a
polymerization solution containing free radical initiator and
disulfide bisacrylamide and optionally containing an acrylamide,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 11
[0038] wherein n and m are each, independently, a positive integer,
thereby forming a solid support comprising immobilized latent thiol
groups.
[0039] The solid support can be a polymer comprising a reactive
functional group, and the method can further comprise the steps of
contacting the solid support with a compound comprising a
functional group which can react to form a bond with the reactive
functional group and a substituted or unsubstituted alkenyl group
or a group having at least one .alpha.,.beta.-unsaturated carbonyl,
thereby forming a solid support having immobilized unsaturated
aliphatic group, and contacting the unsaturated aliphatic groups of
the solid support with a polymerization solution containing free
radical initiator, a disulfide bisacrylamide and optionally
containing an acrylamide, wherein the disulfide bisacrylamide is
represented by the following structural formula: 12
[0040] wherein n and m are each, independently, a positive integer,
thereby forming a solid support comprising immobilized latent thiol
groups.
[0041] The polymeric solid support can be polystyrene. The reactive
functional group of the polymeric solid support can be an amine
group or a hydroxyl group and the compound is represented by the
following structural formula: 13
[0042] wherein Y is a leaving group, L is a linking group, and
R.sub.10 is a substituted or unsubstituted alkenyl group or a group
having at least one .alpha.,.beta.-unsaturated carbonyl. Y can be:
14
[0043] wherein R.sub.6 and R.sub.7 are each, independently, a
substituted or unsubstituted alkyl group, a substituted or
unsubstituted alkenyl group, a substituted or unsubstituted
aromatic group, a substituted or unsubstituted heteroaromatic
group, a substituted or unsubstituted aralkyl, or a substituted or
unsubstituted heteroaralkyl group.
[0044] The method can further comprise the step of derivatizing a
solid support with a latent thiol group, thereby forming a solid
support having immobilized latent thiol groups. The solid support
can be doped or undoped silica, alumina, quartz or glass, and the
solid support can be derivatized by contacting it with a compound
comprising a silane group or a carboxylic acid group and a latent
thiol group.
[0045] The solid support can be platinum or palladium, and the
solid support is derivatized by contacting it with a compound
comprising a nitrile or isonitrile group and a latent thiol
group.
[0046] The solid support can be a polymer comprising reactive
functional groups, and the solid support is derivatized by
contacting it with a compound comprising a functional group which
can react to form a bond with the reactive functional group and a
latent thiol group. The polymeric solid support can be polystyrene.
The reactive functional group of the polymeric solid support can be
an amine or a hydroxyl group and the solid support can be
derivatized by contacting the solid support with a compound
represented by the following structural formula: 15
[0047] wherein Y is a leaving group, L is a linking group, and
R.sub.4 is a thiol protecting group, thereby forming a solid
support having immobilized latent thiol groups. Y can be: 16
[0048] wherein R.sub.6 and R.sub.7 are each, independently, a
substituted or unsubstituted alkyl group, a substituted or
unsubstituted alkenyl group, a substituted or unsubstituted
aromatic group, a substituted or unsubstituted heteroaromatic
group, a substituted or unsubstituted aralkyl, or a substituted or
unsubstituted heteroaralkyl group. R.sub.4 can be: 17
[0049] wherein R.sub.6 is a substituted or unsubstituted alkyl
group, a substituted or unsubstituted alkenyl group, a substituted
or unsubstituted aromatic group, a substituted or unsubstituted
heteroaromatic group, a substituted or unsubstituted aralkyl, or a
substituted or unsubstituted heteroaralkyl group.
[0050] In another embodiment, the invention is directed to a method
of making a solid support having immobilized thiol groups,
comprising the steps of contacting a glass solid support with a
silane compound represented by the following structural formula:
18
[0051] whereinX is a halogen, and R.sub.1, R.sub.2 and R.sub.3 are
each, independently, a halogen, an alkyl group, an alkenyl group or
a group having at least one .alpha.,.beta.-unsaturated carbonyl,
provided that at least one of R.sub.1, R.sub.2 or R.sub.3 is an
alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl, thereby forming a solid
support having an unsaturated aliphatic surface, contacting the
unsaturated aliphatic surface of the solid support with a
polymerization solution containing free radical initiator, a
disulfide bisacrylamide and optionally containing an acrylamide,
wherein the disulfide bisacrylamide is represented by the following
structural formula: 19
[0052] wherein n and m are each, independently, a positive integer,
thereby forming a solid support comprising immobilized latent thiol
groups, and contacting the latent thiol groups with a disulfide
reducing agent, thereby forming a solid support having immobilized
thiol groups.
[0053] In another embodiment, the invention is directed to a method
of forming an array of nucleic acids immobilized on a solid support
including forming an amine-derivatized region on the support,
treating the amine-derivatized region with a thiolating agent such
that latent thiol groups immobilized on the support are formed,
activating the latent thiol groups, contacting the activated thiol
groups with a plurality of nucleic acids comprising acrylamide
functional groups, and forming a covalent bond between the two
groups, thereby forming an array of nucleic acids immobilized on
the solid support. In alternate embodiments, each nucleic acid
contained in the array includes a nucleotide sequence identical to
or substantially identical to, the nucleotide sequence of the other
nucleic acids of the array, or nucleic acids with a plurality of
nucleotide sequences are contained in the array. The solid support
can include a plurality of amine-derivatized regions. The method
can further include a step of blocking any unbonded reactive thiol
groups remaining following the binding of the nucleic acids to the
thiol groups.
[0054] In another aspect, the invention is directed to a kit for
attaching nucleic acids to a solid support including a solid
support component including a plurality of immobilized latent thiol
groups and instructions for activating the thiol groups to form
covalent bonds with nucleic acids including acrylamide functional
groups. Such kits can also include an activator component, an
acrylamide functional nucleic acids component, a blocking component
and/or a wash buffer.
[0055] In an alternate embodiment, the invention is directed to a
kit for attaching nucleic acids to a solid support including a
solid support component including a plurality of immobilized latent
thiol groups and nucleic acids including acrylamide functional
groups. In a particular embodiment, the nucleic acids are
immobilized on the solid support by a covalent bond between the
immobilized thiol groups and the nucleic acids. Such kits can also
include an activator component, a blocking component and/or a wash
buffer.
[0056] In another aspect, the invention is directed to a method for
detecting or separating target nucleic acids from other components
contained in a sample including providing a solid support
comprising a plurality of immobilized nucleic acids comprising
nucleotide sequences complementary to a subsequence of the
nucleotide sequence of the target nucleic acid, wherein the nucleic
acids are immobilized by a covalent bond formed between a thiol
group immobilized on the solid support and an acrylamide functional
group contained on the nucleic acid, contacting the immobilized
nucleic acid with the test sample, and hybridizing target nucleic
acids to immobilized nucleic acids with complementary subsequences,
thereby separating target nucleic acids from other components
contained in the sample. After detection or separation, the target
nucleic acids can be amplified. The method can be used in an assay
for detecting a contaminant in a sample, for medical diagnosis of a
medical condition, for genetic and physical mapping of genomes, for
monitoring gene expression and for DNA sequencing.
[0057] In another embodiment, the invention is directed to a method
for detecting or separating target nucleic acids from other
components contained in a sample including providing a solid
support comprising a plurality of immobilized thiol groups,
contacting the thiol groups with a plurality of nucleic acids
comprising nucleotide sequences complementary to a subsequence of
the nucleotide sequence of the target nucleic acid and acrylamide
functional groups, forming a covalent bond between the two groups,
thereby immobilizing the nucleic acids on the solid support,
contacting the immobilized nucleic acids with the test sample, and
hybridizing target nucleic acids to immobilized nucleic acids with
complementary subsequences, thereby detecting or separating target
nucleic acids from other components contained in the sample. After
detection or separation, the target nucleic acids can be amplified.
The method can be used in an assay for detecting a contaminant in a
sample, for medical diagnosis of a medical condition, for genetic
and physical mapping of genomes, for monitoring gene expression and
for DNA sequencing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1 is a schematic representation of a method of
derivatizing an amine group bearing polystyrene support with an
acrylamide derivatized oligonucleotide.
[0059] FIG. 2A is a representation of a solid support selectively
treated to activate latent thiol groups.
[0060] FIG. 2B is a representation of a solid support in which
reactive groups are blocked, then exposed to acrylamide functional
oligonucleotides.
[0061] FIG. 3A is a photograph showing results of microarray
formation on a N,N' bis(acryloyl)cystamine (BAC) coated slide in
which excess thiol groups were blocked with dimethylacrylamide
(DMA).
[0062] FIG. 3B is a photograph showing results of microarray
formation on a N,N' bis(acryloyl)cystamine (BAC) coated slide in
which excess thiol groups were blocked with 2-hydroxymethacrylate
(HEMA).
[0063] FIG. 4 is a plot of fluorescent intensity across a spot for
slides prepared with 2% N,N' bis(acryloyl)cystamine (BAC) or 2%
N,N' bis(acryloyl)cystamine(BAC) plus P400 mm.
[0064] FIGS. 5A-5C are photographs showing a N,N'
bis(acryloyl)cystamine (BAC) acrylate slide after hybridization to
fluorescent complementary oligonucleotide probes.
[0065] FIG. 6 is a bar graph illustrating the results obtained from
a comparison of hybridizations performed using Tris-Glycine buffer
and those performed using carbonate buffer.
[0066] FIG. 7 is a bar graph illustrating the results of an
experiment showing the influence of buffer and glycerol in spotting
solution on hybridization signal for 10 .mu.l probe spots.
[0067] FIG. 8 is a schematic of a synthesis for a non-symmetrical
disulfide acrylamide.
[0068] FIG. 9 is a schematic of a synthesis for a non-symmetrical
disulfide acrylamide.
DETAILED DESCRIPTION OF THE INVENTION
[0069] The present invention is based, at least in part, on the
discovery of a novel and convenient method of immobilizing an
affinity ligand on a solid support. The method utilizes a covalent
bond formed between a thiol group immobilized on the solid support
and an acrylamide functional group contained on an affinity ligand
to immobilize the affinity ligand to the support. In a particular
embodiment, the covalent bond formed is a sulfide, a thioether,
bond.
[0070] The method and the supports it produces are advantageous in
several respects. The method utilizes reagents which are both
readily available and compatible with the types of analysis
conducted with solid supports. Because the materials can be used in
aqueous solutions, the need for special skills and sophisticated
chemical apparatus are minimized. In addition, because the
materials and the supports they form are quite stable, the
reproducibility from support to support which has previously proved
so difficult to achieve can be realized. This stability also
permits the components forming the bond to be combined at different
times. For example, because solid supports containing the latent
thiol groups of the invention are extremely stable, they can be
produced under consistent conditions for use at a later time. Prior
to analysis, the latent thiol groups can be activated and contacted
with the acrylamide modified nucleic acids to form a support
containing immobilized nucleic acids. In a particular embodiment,
the thiol groups are activated by contact with a reducing
agent.
[0071] In one embodiment, the method is directed to a method of
immobilizing an affinity ligand on a solid support. The method
includes providing a solid support comprising an immobilized thiol
group, contacting the thiol group with an affinity ligand
comprising an acrylamide functional group, and forming a covalent
bond between the two groups, thereby immobilizing the affinity
ligand on the solid support.
[0072] The term "affinity ligand" is intended to include any
molecule that can form a specific binding complex with a target
analyte and can be immobilized on a suitable solid support. Any
suitable ligand can be used in the present invention provided that
it can form a specific binding complex with a target analyte.
Methods for determining the thermal stability of binding complexes
and, in particular, hybridization complexes are well known in the
literature. Wetmur, Critical Reviews in Biochemistry and Molecular
Biology, 26:227-259 (1991); Quartin and Wetmur, Biochemistry,
28:1040-1047 (1989).
[0073] One especially useful example of an affinity ligand is a
single-stranded nucleic acid, which can bind by hybridization, for
example, to an analyte that contains a complementary nucleic acid
sequence. The single stranded nucleic acid affinity ligand can be
complementary to the entire analyte nucleic acid sequence or to a
portion thereof. Single-stranded nucleic acids can also be used for
isolation of duplex nucleic acids by triplex formation (Hogan and
Kessler, U.S. Pat. No. 5,176,966 and Cantor, et al., U.S. Pat. No.
5,482,836, the teachings of which are incorporated herein by
reference). Double-stranded nucleic acids can also serve as useful
affinity ligands for nucleic acid binding proteins, or for nucleic
acid analytes that bind to the ligand by triplex or tetraplex
formation. The conditions under which a single stranded nucleic
acid will bind to another nucleic acid to be immobilized on a solid
support can be estimated by those skilled in the art using the
procedure referenced above. In addition, the melting temperature
(T.sub.m) of the two nucleic acids provides a reasonable framework
for estimating the temperate at which an nucleic acid analyte will
hybridize to a nucleic acid affinity ligand. In general, the
T.sub.d is lower than the T.sub.m by about 15 to 25.degree. C. and,
therefore, the temperature at which the gel should be run to
facilitate specific hybridization between the analyte and affinity
ligand should be about 15 to 25.degree. C. or more below the
T.sub.m.
[0074] Nucleic acid aptamers (Tuerk and Gold, Science (1990)
249:5050; Joyce, Gene (1989), 82:83-87; Ellington and Szostak,
Nature (1990), 346:818-822) can also be used as affinity ligands in
the process of the present invention. Aptamers can be selected
against many kinds of analytes, including proteins, small organic
molecules, and carbohydrates (reviewed in Klug and Famulok,
Molecular Biology Reports (1994), 20:97-107). Thus, selection of
aptamer ligands offers a simple and flexible mechanism for
obtaining affinity ligands against virtually any target
molecule.
[0075] Other useful ligands include proteins or polypeptides which
can bind to specific analytes. An especially useful class of
protein ligands are antibody molecules, which can be elicited
against a wide range of analytes by immunization methods.
Antibodies ligands can be monoclonal or polyclonal. In addition, a
fragment of an antibody can be an affinity ligand. Similarly,
receptor proteins may be useful as ligands for purification and
detection of analytes that bind to or are bound by them.
[0076] Carbohydrates have been successfully used as affinity
ligands for electrophoretic purification of lectins (Horejsi and
Kocourek, Biochim. Biophys. Acta (1974), 336:338-343), and may be
useful for purification and detection of molecules that bind to
specific carbohydrates or glycoproteins.
[0077] Binding or non-binding conditions of proteins, aptamers and
lectins for specific ligands can be estimated using the procedure
outlined above for estimating the stability of analyte/affinity
ligand complexes. In addition, equilibrium dialysis experiments can
provide a rational method of predicting the stability of
analyte/affinity ligand complexes. For example, the dissociation
constant of a protein for a particular ligand can be determined in
the electrophoresis buffer at several different pHs, temperatures
or ionic strengths. The higher the dissociation constant, the
weaker the binding between the protein and the ligand (see Segel,
I. H., Biochemical Calculations, 2.sup.nd Edition (1976), John
Wiley & Sons, New York, p. 241-244). From this data a binding
and a non-binding condition can be estimated.
[0078] Many other types of immobilized ligands are possible
including peptides, amino acids, nucleosides, small organic
molecules, lipids, hormones, drugs, enzyme substrates, enzyme
inhibitors, enzymes, coenzymes, inorganic molecules, chelating
agents, macromolecular complexes, polysaccharides, monosaccharides,
and others.
[0079] In a particular embodiment, a nucleic acid can be utilized
as an affinity ligand. Such nucleic acids include deoxyribonucleic
acid (hereinafter "DNA"), or ribonucleic acid hereinafter "RNA"),
modified nucleic acids, nucleic acid analogs, and chimeric
molecules of a mixed class comprising a nucleic acid with another
organic component, e.g., peptide nucleic acids. Nucleic acids can
be single-stranded or double-stranded nucleic acids. Typically, the
length of a nucleic acid will be at least about 5 nucleotides in
length, more typically between about 5 and 100 nucleotides even
more typically between 5 and 50, although it can be as long as
several thousand bases.
[0080] Such nucleic acids are typically "isolated" nucleic acids,
e.g., nucleic acids separated away from the components of their
source of origin (e.g., as it exists in cells, or in a mixture such
as a library) and can have undergone further processing. Isolated
nucleic acids include nucleic acids obtained by methods known to
those of skill in the art. These isolated nucleic acids include
substantially pure nucleic acids, e.g., nucleic acids free from
protein, carbohydrate or lipids. Nucleic acids can be produced by
chemical synthesis, or by combinations of biological and chemical
methods or by recombinant methods.
[0081] The term "modified nucleic acid" is intended to include
nucleic acids containing modified bases, deoxyribose groups or
phosphates. Examples of nucleic acids having modified bases,
include, for example, acetylated, carboxylated or methylated bases
e.g, 4-acetylcytidine, 5-carboxymethylaminomethyluridine,
1-methylinosine, norvaline or allo-isoleucine.
[0082] The term "nucleic acid analog" is intended to include
molecules that lack a conventional
deoxyribose/ribose-phosphodiester backbone, but which retain the
ability to form Watson-Crick type base pairs with complementary
single-stranded nucleic acids. Examples of nucleic acid analogues
include peptide nucleic acids (PNAS; Egholm et al., 1992, J. Am.
Chem. Soc. 114: 1895-1897) and morpholino oligomers (morpholinos;
Summerton and Weller, Antisense Nucleic Acid Drug Dev.,
(1997)7:187-195). It will be apparent to those skilled in the art
that similar design strategies can be used to construct other
nucleic acid analogs that will have useful properties for
immobilized probe assays.
[0083] The term "alkyl group", as used herein, is intended to
include straight chained or branched C.sub.1-C.sub.18 hydrocarbons
which are completely saturated, or cyclic C.sub.3-C.sub.18
hydrocarbons which are completely saturated. Lower alkyl groups are
straight chained or branched C.sub.1-C.sub.3 hydrocarbons or
C.sub.3-C.sub.8 cyclic hydrocarbons which are completely saturated.
Alkyl groups are preferably lower alkyl groups.
[0084] The term "alkenyl group," as used herein, is intended to
include straight chained or branched C.sub.1-C.sub.18 hydrocarbons
which have one or more double bond, or cyclic C.sub.3-C.sub.18
hydrocarbons which have one or more unconjugated double bond. Lower
alkenyl groups are straight chained or branched C.sub.1-C.sub.8
hydrocarbons which have one or more double bond or C.sub.3-C.sub.8
cyclic hydrocarbons which have one or more unconjugated double
bond. Alkenyl groups are preferably lower alkenyl groups.
[0085] The term "aromatic group" is intended to include carbocyclic
aromatic ring systems (e.g., phenyl) and carbocyclic aromatic ring
systems fused to one or more carbocyclic aromatic or non-aromatic
ring (e.g., naphthyl, anthracenyl and
1,2,3,4-tetrahydronaphthyl).
[0086] Heteroaromatic groups, as used herein, include heteroaryl
ring systems (e.g., thienyl, pyridyl, pyrazole, isoxazolyl,
thiadiazolyl, oxadiazolyl, indazolyl, furans, pyrroles, imidazoles,
pyrazoles, triazoles, pyrimidines, pyrazines, thiazoles,
isoxazoles, isothiazoles, tetrazoles, or oxadiazoles) and
heteroaryl ring systems in which a carbocyclic aromatic ring,
carbocyclic non-aromatic ring, heteroaryl ring or a
heterocycloalkyl ring is fused to one or more other heteroaryl
rings (e.g., benzo(b)thienyl, benzimidazole, indole,
tetrahydroindole, azaindole, indazole, quinoline, imidazopyridine,
purine, pyrrolo[2,3-d]pyrimidine, and pyrazolo [3
,4-d]pyrimidine).
[0087] The term "aralkyl group," as used herein, is intended to
include aromatic substituents that are linked to a moiety by an
alkyl group that preferably has from one to about six carbon
atoms.
[0088] The term "heteroaralkyl group," as used herein, is intended
to include heteroaromatic substituents that are linked to a moiety
by an alkyl group that preferably has from one to about six carbon
atoms.
[0089] The term "heterocycloalkyl group," as used herein, is
intended to include non-aromatic ring systems that preferably has 5
to 6 atoms and include at least one heteroatom, such as nitrogen,
oxygen, or sulfur. Examples of heterocycloalkyl groups include
morpholines, piperidines, and piperazines.
[0090] Suitable substituents for aliphatic groups, aromatic groups,
aralkyl groups, heteroaromatic groups and heterocycloalkyl groups
include aromatic groups, halogenated aromatic groups, lower alkyl
groups, halogenated lower alkyl (e.g. trifluoromethyl and
trichloromethyl), --O-(aliphatic group or substituted aliphatic
group), --O-(aromatic group or substituted aromatic group), benzyl,
substituted benzyl, halogens, cyano, nitro, --S-(aliphatic or
substituted aliphatic group), and --S-(aromatic or substituted
aromatic).
[0091] The term "linking group," as used herein, includes
substituted or substituted alkyl groups, substituted or
unsubstituted aromatic groups, substituted or unsubstituted aralkyl
groups and substituted or unsubstituted polyether groups.
[0092] The affinity ligands of the invention comprise a
.alpha.,.beta.-unsaturated carbonyl group. A preferred
.alpha.,.beta.-unsaturated carbonyl group is an acrylamide. The
term "acrylamide" is intended to include compounds represented by
Structural Formula IV: 20
[0093] In Structural Formula IV, R.sub.11 is --H, or a substituted
or unsubstituted alkyl group. In a preferred embodiment, R.sub.11
is a --H or a methyl group.
[0094] An affinity ligand can be derivatized with a selectively
thiol reactive group. Such thiol reactive groups can include
methacrylate, methacrylamide, .alpha.,.beta. unsaturated carbonyl
groups [CH2CHC(F2)], .alpha.,.beta. unsaturated difluoro groups and
maleimide groups. In general, such groups show enhanced reactivity
with thiol groups, as opposed to other functional gorups present in
the reaction.
[0095] In several embodiments of the invention, it is useful to
provide latent thiol groups through the use of polymerizable
disulfide compounds. As indicated in Structures IIB-D, such
compounds can be monofunctional or bifunctional with regard to the
.alpha.,62 unsaturated carbonyl group. A commercially available
example of a bifunctional disulfide reagent is BAC. An example of a
monofunctional disulfide reagent is AEMA (Schnaar, R. L. et al.,
1985, Analytical Biochemistry, 151:268-281). Additional
monofunctional acrylamide disulfide derivatives can be generated by
reacting BAC with the reducting agents .beta.-mercaptoethanol and
thioacetic acid, as shown if FIGS. 8 and 9.
[0096] The term "acrylamide group" is intended to include those
groups which are represented by Structural Formula V: 21
[0097] In Structural Formula V, R.sub.11 is defined as in
Structural Formula IV. "}" represents the point of attachment of
the affinity ligand. Methods for derivatizing nucleic acid affinity
ligands with an acrylamide group can be found in Boles, et al.,
U.S. Pat. No. 5,932,711 and Hoffman and Dong, U.S. Pat. No.
5,034,428, the entire teachings of which are incorporated herein by
reference.
[0098] A peptide or protein can be derivatized with an acrylamide
group by reacting an amine group with an acrylic acid in the
presence of a coupling agent such as dicyclohexylcarbodiimide or
diisopropylcarbodiimide. The amine group of the peptide or protein
can react with the acrylic acid to form an acrylamide group
represented by Structural Formula V. Methods for coupling peptide
or protein amine groups with carboxylic acid group, such as the
carboxylic acid group of an acrylic acid, can be found in Stewart
and Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce
Chemical Company, Rockford, Ill., the entire teachings of which are
hereby incorporated by reference.
[0099] Carbohydrates, antigens or drug molecules which have an
amine group can also be coupled with acrylic acid to form an
acrylamide group using a coupling agent such as
dicyclohexylcarbodiimide or diisopropylcarbodiimide. Alternatively,
the carboxylic acid group of acrylic acid can be converted into an
active ester, such as a p-nitrophenol acrylate, a o,p-dinitrophenol
acrylate, or N-hydroxysuccinamide acrylate, and then allowed to
react with an amine group of a carbohydrate, antigen or drug
molecule.
[0100] A thiol group is a group of the formula --SH. The term
"latent thiol group" is intended to include thiol groups which have
been protected with a thiol protecting group and disulfide groups
of a polymer matrix. The term "thiol protecting group" is intended
to include groups which can react with a thiol group causing the
thiol group to be unreactive and which can be removed to regenerate
the thiol group. Thiol protecting groups are known to those skilled
in the art. For examples of thiol protecting groups see Greene, et
al., Protective Groups in Organic Synthesis (1991), John Wiley
& Sons, Inc., pages 277-308, the teachings of which are
incorporated herein by reference in their entirety. In one
embodiment, thiol protecting groups can include the following
groups: 22
[0101] The term "Acrydite.TM. phosphoramidite" as used herein
refers to the proprietary acrylamide phosphoranidite sold by Mosaic
Technologies, Waltham, Mass. This product allows addition of an
acrylamide group directly to a DNA or an RNA oligonucleotide using
standard beta-cyanoethylphosphoramidite methods.
[0102] The acronym "AEMA" is intended to encompass the compound
known as
4-[[1-Oxo-3-[[2-[(1-oxo-2-propenyl)-amino]ethyl]dithio]propyl]amino]butan-
oic acid which was obtained from Ronald L. Schnaar, Department of
Pharmacology and Neuroscience, The Johns Hopkins University School
of Medicine, Baltimore, Md. (Schnarr, R. L. et al., 1985 Analytical
Biochemistry 151:268-281).
[0103] The acronym "APS" is intended to encompass an ammonium
persulfate such as that available from BioRad Laboratories, Inc.,
Hercules, Calif.
[0104] The term "acrylate slide" is intended to encompass a slide,
e.g, a lass microscope slide, coated with an organosilane compound
that includes an acrylamide or acrylic ester functionality. Such
slides can be generated by treatment with
(3'-acryloxypropyl)trimethoxysilane or other similar compounds
available commercially, for example, from Gelest, Tullytown, Pa.
Such slides can also be commercially obtained for example, from CEL
Associates, Inc., Houston, Tex., (see Cat.# ACR-25C).
[0105] The acronym "BAC" is intended to encompass the compound
known as N,N'-bis(acryloyl)cystamine available, for example, from
Fluka; Buchs, Switzerland.
[0106] The acronym "DMA" is intended to encompass the compound
known as dimethylacrylamide.
[0107] The acronym "DMSO" is intended to encompass the compound
known as dimethyl sulfoxide.
[0108] The acronym "DTNB" is intended to encompass the compound
known as 5,5'-dithio-bis-(2-nitrobenzoic) acid.
[0109] The acronym "HEMA" is intended to encompass the compound
known as 2-hydroxymethacrylate.
[0110] The acronym "ME" is intended to encompass compounds known as
mercaptoethanol.
[0111] The acronym "P400 mm" is intended to encompass compounds
known as pol(yethylene glycol) 400 monomethyl ether
monomethacrylate.
[0112] The acronym "SATP is intended to encompass the compound
known as N-succinimidyl S-acetylthiopropionate available, for
example, from Pierce; Rockford, Ill.
[0113] The acronym "SBB" is intended to encompass sodium borate
buffers.
[0114] The acronym "SDS" is intended to encompass the compound
known as sodium dodecyl sulfate.
[0115] The acronym "SSPE" is intended to encompass standard saline
phosphate EDTA buffers.
[0116] The acronym "TAA" is intended to encompass thioacetic
acids.
[0117] The acronym "TCEP" is intended to encompass the compound
known as tris(2-carboxyethyl) phosphine hydrochloride.
[0118] The term "TE buffer" is intended to encompass a 10 mM
Tris-HCl pH 8.3; 1 mM EDTA buffer.
[0119] The acronym "TEMED" is intended to encompass compounds known
as N,N,N',N'-tetra-methyl-ethylenediamine available, for example,
from BioRad Laboratories, Inc., Hercules,
[0120] The term "GMS spotter" is intended to include a "GMS 417
Arrayer" (Affymetrix; Santa Clara, Calif.).
[0121] In a preferred embodiment, the thiol protecting group is a
disulfide group. Disulfide protecting groups can be removed by
treating with a disulfide reducing agent which reduces the
disulfide bond to form two thiol groups. Disulfide reducing agents
include compounds such as tris(2-carboxyethyl)phosphine
hydrochloride (TCEP), .beta.-mercaptoethanol and
dithiothreitol.
[0122] A solid support having immobilized thiol groups is contacted
with an affinity ligand of the invention, which has been
derivatized with an acrylamide group. The thiol groups can react
with the acrylamide group of the affinity ligand to form a covalent
bond via a Michael condensation reaction to form a solid support
having immobilized affinity ligands. Therefore, although the term
"immobilized" when used in reference to other methods can encompass
various means of attachment to a solid support including both ionic
and covalent types of bonding, when used in reference to the
present invention "immobilized" refers to attachment with a
covalent bond.
[0123] The solid supports of the invention can be formed from a
variety of materials including paper, glass, silica, metals,
ceramics, plastic and polymers. Polymers can be cross-linked to
form gels, e.g., electrophoretic gels, e.g., acrylamide gels. The
solid supports can be of any shape or dimension. Porous filters,
woven materials and meshes, planar sheets, microparticles, fibers,
rods, optical fibers, dipsticks, beads, tubes, multiwell plates,
cups and capillaries can all be used as solid supports.
[0124] In a preferred embodiment, the solid support of the
invention is formed of glass, silica, metal, ceramic or a polymer
such as polystyrene, crosslinked polystyrene, polyethylene,
polypropylene, polyrmethacrylate, dextran and agarose and a polymer
layer is applied to a surface of the solid support. In particularly
preferred embodiments, the solid support is formed of glass and a
polymer layer is applied to a surface of the solid support. In a
particularly preferred embodiment, the solid support is planar in
form and contains a polymer layer applied to a surface.
[0125] A preferred embodiment when the solid support is a
chromotography bead, e.g., a polyacrylamide bead, is the use of BAC
to form the thiol groups.
[0126] In one embodiment, an aliphatic group having a substituted
or unsubstituted alkenyl group or a .alpha.,.beta.-unsaturated
carbonyl group is attached to a surface by contacting the surface
with an aliphatic group which has been derivatized with a group
that can bind to the surface, thereby forming an unsaturated
aliphatic surface. Therefore, selection of a functional group with
which the aliphatic group is to be derivatized is dependent on the
type of material to which the aliphatic group is to be attached.
When the surface to which the aliphatic group is to be attached is
doped or undoped silica, alumina, quartz or glass, the aliphatic
group is preferably derivatized with a silane group or carboxylic
acid. In on embodiment, when the aliphatic group is derivatized
with a silane group, the compound can be represented by Structural
Formula I.
[0127] In one embodiment, a glass or silica support is treated with
an appropriate organosilane compound to provide a surface layer
comprising a plurality of alpha-beta unsaturated groups. Preferred
silanes include alkoxysilanes and chlorosilanes having vinyl,
allylic, acrylamide, methacrylamide or acrylic ester
functionalities. One preferred silane is
(3'-acryloxpropyl)trimethoxysilane. This and other preferred
silanes are commercially available from, for example, Gelest
(Tullytown, Pa.).
[0128] When the aliphatic group is to be attached to a surface
which is gold, silver, copper, cadmium, zinc, palladium, platinum,
mercury lead, iron, chromium, manganese, tungsten, or any alloys of
the above metals, the aliphatic group to be attached is preferably
derivatized with a thiol, sulfide or disulfide group. When the
surface to which the aliphatic group is to be attached is platinum
or palladium, the aliphatic group is preferably derivatized with a
nitrile or isonitrile group. Finally, when the surface to which the
aliphatic group is to be attached is copper, the aliphatic group is
preferably derivatized with a hydroxamic acid group.
[0129] An acrylamide gel having latent thiol groups can be formed
on the unsaturated aliphatic surface of the solid support by
contacting the unsaturated aliphatic surface with a polymerization
solution containing a free radical initiator, a disulfide
bisacrylamide and optionally containing an acrylamide. Conditions
for free radical polymerization of disulfide bisacrylamide monomers
are similar to those used for polymerization of acrylamide monomers
(for example, see Perbal, A Practical Guide to Molecular Cloning,
2nd Edition, (1988), John Wiley & Sons, New York, pages 15-17)
and are further described in Example 5 and Example 7. Typically,
the polymerization solution contains a disulfide bisacrylamide in
about 0.1% to about 20% in an aqueous solution. If an acrylamide
and/or a bisalkylene acrylamide is also present, the concentration
of the disulfide bisacrylamide and the acrylamide and/or the
bisalkylene acrylamide together is about 0.1% to about 20%.
Optionally, an organic solvent, such as DMF, can be used to improve
reactivity and/or solubility. The polymerization reaction is
initiated by a free radical initiator. A free radical initiator is
a substance which decomposes to form a free radical. Typical free
radical initiators include ammonium persulfate, peroxides, and azo
compounds such as azobisisobytyronitrile. Ammonium persulfate is a
preferred free radical initiator. About 0.1% (weight/volume) to
about 10% (weight/volume) of the free radical initiator is added to
the polymerization solution either before the solution is in
contact with the unsaturated aliphatic surface or after the
polymerization solution is in contact with the unsaturated
aliphatic surface.
[0130] Polymerization of the disulfide bisacrylamide on the surface
of the solid support forms a solid support having immobilized
disulfide groups which are latent thiol groups. The immobilized
latent thiol groups can be converted to immobilized thiol groups by
contacting the solid support with a disulfide reducing agent such
as tris(2-carboxyethyl)phosphine hydrochloride (TCEP),
.beta.-mercaptoethanol and dithiothreitol.
[0131] Comonomers can be added to the BAC for co-polymerization.
Useful comonomers include for example, acrylamide, bis acrylamide;
N,N-dimethyl acrylamide, N-octyl acrylamide, poly(ethylene glycol)
(n) dimethacrylate, n 200 or 400, (Catalog # 00096 and 02364
(1998-2000 "Polymers and Monomers" Catalog, Polysciences, Inc,
Warrington Pa.)). A preferred comonomer is pol(yethylene glycol)
400 monomethyl ether monomethacrylate (P400 mm, Catalog # 16665
(1998-2000 "Polymers and Monomers" Catalog, Polysciences, Inc,
Warrington Pa.)).
[0132] Other comonomers that could be, used are well known to those
practiced in the art of polymer science and coatings; (see, e.g,
1998-2000 "Polymers and Monomers" Catalog, Polysciences, Inc,
Warrington Pa.) In addition, it is well known that mixtures of
three or more comonomers can be mixed to achieve polymers with
desired properties. Comonomers can be added in organic solvents.
Optionally, an organic solvent, such as DMF can be used improve
reactivity and/or solubility.
[0133] In an alternate embodiment, a solution of acrylamide and
non-symmetrical disulfide acrylamides are prepared together with a
crosslinking compound such as bis-acrylamide. The mixture is
polymerized using ammonium persulfate with TEMED, ultraviolet (UV)
light, heat, ionizing radiation or or an equivalent known to those
of skill in the art. The disulfide bonds are reduced, for example,
using TCEP or a thiol exchange reaction with DTT. Thin polymer
layers can be produced by dipping slides in a polymerizing
solution. Thicker gels can be formed between glass plates.
[0134] In another embodiment, the solid support is a polymer which
has reactive functional groups. Reactive functional groups include
amines, amides, hydroxyl, carboxylic acid, and halogens. A
preferred polymeric solid support is a polystyrene which has
reactive functional soups. Preferred reactive functional groups are
amine and hydroxyl groups. The solid support is contacted with a
compound which has a functional group which can react with the
reactive functional group of the polymer to form a double bond and
a substituted or unsubstituted alkenyl or at least one
.alpha.,.beta.-unsaturated carbonyl to form a solid support having
unsaturated aliphatic groups. When the reactive functional group is
a halogen, it can react, for example, with an amine or an alkoxide
to form a covalent bond. When the reactive functional group is a
carboxylic acid, it can react, for example, with an amine or a
hydroxide in the presence of dicyclohexylcarbodiimide. When the
reactive functional group is an amine or a hydroxyl group, it can
react, for example, with an ester, a carboxylic acid or a halogen
to form a covalent bond. In a preferred embodiment when the solid
support has an amine or a hydroxyl reactive group, it is contacted
with a compound is represented by Structural Formula VI: 23
[0135] In Structural Formula VI, Y and L are as defined in
Structural Formula III, and R.sub.10 is a substituted or
unsubstituted alkenyl group or a group having at least one
.alpha.,.beta.-unsaturated carbonyl. The immobilized unsaturated
aliphatic groups are then contacted with a polymerization solution
containing a free radical initiator, a disulfide bisacrylamide and
optionally containing an acrylamide to form a solid support having
immobilized latent thiol groups in an acrylamide gel. The latent
thiol groups can be activated by contacting the gel with a
disulfide reducing agent.
[0136] In another embodiment, the polymeric solid support which is
functionalized with an amine or hydroxyl reactive functional groups
is reacted with a compound represented by Structural Formula III to
form a solid support having immobilized latent thiol groups. In
this embodiment, the solid support is preferably, cellulose,
celite, poly(acrylic acid), polystyrene, cross-linked polystyrene,
an agarose or cross-linked agarose, such as Sepharose or Superose,
a cross-linked dextran, such as Sephadex or Sephacryl, or a
composite of cross-linked agarose and dextran, such as Superdex.
The latent thiol groups are activated by removing the thiol
protecting groups. Methods for removing thiol protecting groups can
be found in Greene, et al., Protective Groups in Organic Synthesis
(1991), John Wiley & Sons, Inc., pages 277-308, the teachings
of which are incorporated herein by reference in their
entirety.
[0137] In another embodiment, the solid support is silica, alumina,
quartz or glass, and the solid support is derivatized with a latent
thiol group by contacting the solid support with a compound which
has a silane group or a carboxylic acid group and a latent thiol
group. In a preferred embodiment, the compound can be represented
by Structural Formula VII: 24
[0138] In Structural Formula VII, R.sub.4 and L are defined as
above, and Z is a carboxylic acid group or a silane group of the
formula: 25
[0139] wherein R.sub.11, R.sub.12 and R.sub.13 are each,
independently, a halogen, a substituted or unsubstituted alkyl
group, a substituted or unsubstituted aromatic group or a
substituted or aralkyl group, provided that at least one of
R.sub.11, R.sub.12 or R.sub.13 is a halogen. "{" represents the
attachment of the silane group to the linking group represented by
"L".
[0140] In another embodiment, the solid support is platinum or
palladium, and the solid support is derivatized with a latent thiol
group by contacting the solid support with a compound that has a
nitrile or an isonitrile group and a latent thiol group. In a
preferred embodiment, the compound can be represented by Structural
Formula VIII: 26
[0141] In Structural Formula VIII, R.sub.4 and L are defined as
above, and Z.sub.1 is a nitrile or an isontrile group.
[0142] In another embodiment, the solid support is copper and the
solid support is derivatized with a latent thiol group by
contacting the solid support with a compound that has a hydroxamic
acid group and a latent thiol group.
[0143] The nucleic acids can be immobilized on the surface of the
support in any pattern or arrangement, e.g., blocks, lines, grids
or whorls. Nucleic acids with identical nucleotide sequences can be
immobilized on the solid support, nucleic acids with non-identical
or different nucleotide sequences can be immobilized on the solid
support, and combinations of nucleic acids which contain some
portion with identical nucleotide sequences and some portions which
contain non-identical sequences can be immobilized on the surface
of the solid support.
[0144] In particular embodiments, a plurality of nucleic acids,
portions of which contain identical nucelotide sequences and
portions of which contain non-identical nucleotide sequences, are
attached to the solid support in a manner such that nucleic acids
with non-identical nucleotide sequences are found on spatially
distinct regions of the surface. The phrase "spatially distinct
region" is intended to include a region on the surface of a solid
support around which an imaginary perimeter can be drawn which does
not overlap with the perimeter of any other region.
[0145] The term "array" is intended to include a solid support
containing nucleic acids immobilized on at least one spatially
distinct region of its surface. An array can contain any number of
nucleic acids immobilized within any number of spacially distinct
regions. The spacing and orientation of the nucleic acids can be
regular, e.g., in a rectangular or hexagonal grid, or the pattern
can be irregular or random. In a particular embodiment, nucleic
acids containing non-identical nucleotide sequences are arranged in
a regular pattern on the surface of a solid support. Such an
embodiment is particularly useful, for example, in determining
whether a particular set of components are present in a sample.
Nucleic acids capable of detecting the presence of each component
of the set can be placed in a spacially distinct region, so that in
a single analysis, a determination can be made as to whether one or
more of the components of the set are contained within the sample.
The term "microarray" is intended to include an array in which the
spacially distinct regions containing nucleic acids are relatively
small.
[0146] An affinity ligand having a thiol reactive group may be
contacted with a solid support having free diols either by
immersing the solid support in a solution of ligand, or by
contacting a drop of ligand to the support. In the latter case, the
ligand may be deposited by mechanical contact, as with a metal pin,
or the ligand may be sprayed, as with a piezoelectric dispenser.
When the ligand is deposited onto the surface with a pin or
piezoelectric dispenser, the volume of solution containing the
ligand will vary, depending on the conditions used. For example,
with the Affymetrix Model 417 pin-loop spotter, the volume
deposited depends on the diameter of the loop (see S. Rose,
"Applications of a Novel Microarraying System in Genomics Research
and Drug Discovery, Journal of Association for Laboratory
Automation, 3:(3) 1998) and is in the range of nanoliters (nL) to
picoliters (pL).
[0147] The term "sample" or "test sample" are intended to include
component mixtures which can contain the target molecule. The test
sample can be used directly as obtained from the source or
following pretreatment. The test sample can be derived from any
biological source, such as a physiological fluid, including, blood,
saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine,
peritoneal fluid, amniotic fluid and the like, and fermentation
broths, cell cultures, and chemical reaction mixtures and the like.
The test sample can be pretreated prior to use, such as preparing
plasma from blood, diluting viscous fluids, and the like. Methods
of treatment can involve filtration, distillation, extraction,
concentration, inactivation of interfering components, and the
addition of reagents. In addition, a solid material such as cells
which can contain the target molecule can be used as the test
sample. In some instances, it may be beneficial to modify a solid
test sample to form a liquid medium or to release a target
molecule.
[0148] The solid supports formed by the methods of the invention
can be utilized in a variety of assays. Typically, such assays
include a hybridization reaction between the immobilized nucleic
acid and a target molecule introduced to the solid support, e.g.,
contained in a test sample. It is clear to one of skill in the art
that such methods can be carried out under a range of hybridization
conditions utilizing wash conditions with low to high stringencies.
Conditions can be selected based on the amount of similarity or
differences between the nucleic acids.
[0149] "Stringency conditions" for hybridization is a term of art
which refers to the conditions of temperature and buffer
concentration (ionic strength) which permit hybridization of a
particular nucleic acid to a second nucleic acid in which the first
nucleic acid may be perfectly complementary to the second, or the
first and second may share some degree of complementarity which is
less than perfect. For example, certain high stringency conditions
can be used which distinguish perfectly complementary nucleic acids
from those of less complementarity. "High stringency conditions"
and "moderate stringency conditions" for nucleic acid
hybridizations are explained on pages 2.10.1-2.10.16 (see
particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in
Molecular Biology (Ausubel, F. M. et al., eds., Vol. 1, containing
supplements up through Supplement 29, 1995), the teachings of which
are hereby incorporated by reference. The exact conditions which
determine the stringency of hybridization depend not only on ionic
strength, temperature and the concentration of denaturants such as
formamide or urea, but also on factors such as the length of the
nucleic acid sequence, base composition, percent mismatch between
hybridizing sequences and the frequency of occurrence of subsets of
that sequence within other non-identical sequences. Thus, high or
moderate stringency conditions can be determined empirically.
[0150] By varying hybridization conditions from a level of
stringency at which no hybridization occurs to a level at which
hybridization is first observed, conditions which will allow a
given sequence to hybridize (e.g., selectively) with the most
similar sequences in the sample can be determined. Binding
conditions for triplexes and tetraplexes can be estimated in a
similar manner. A general description of stringency for
hybridization and wash conditions is provided by Ausubel, F. M. et
al., Current Protocols in Molecular Biology, Greene Publishing
Assoc. and Wiley-Interscience 1987, & Supp. 49, 2000, the
teachings of which are incorporated herein by reference. Factors
such as probe length, base composition, percent mismatch between
the hybridizing sequences, temperature and ionic strength influence
the stability of nucleic acid hybrids. Thus, stringency conditions
sufficient to allow hybridization of nucleic acids, can vary
significantly. Such conditions can readily be determined by one of
ordinary skill in the art.
[0151] Such hybridization reactions take place between nucleotide
sequences which are substantially complementary. The phrase
"substantially complementary" is intended to include nucleic acid
sequences which are sufficiently complementary to hybridize with
each other under specified conditions. Typically, complementary
nucleic acids contain at least one complementary subsequence. The
term "subsequence" is intended to include any contiguous segment of
a larger sequence. Thus, a complementary subsequence includes at
least one contiguous segment complementary to the nucleotide
sequence of another nucleic acid.
[0152] Target molecules separated or detected in the assays of the
invention can be amplified. The term "amplified" is intended to
include primer dependent nucleic acid synthesis catalyzed by a
nucleic acid polymerase. For example, the polymerase chain reaction
or hereinafter "PCR" can be utilized to amplify a target molecule.
The method can be used in an assay for detecting a contaminant in a
sample, for medical diagnosis of a medical condition, for genetic
and physical mapping of genomes, for monitoring gene expression and
for DNA sequencing.
[0153] The solid supports formed by the methods of the invention
can be provided in the form of kits. Such kits can contain various
components. In one embodiment, a kit can contain a solid support
containing a plurality of latent thiol groups. Such a kit can be
provided with instructions teaching the purchaser methods for
activating the latent thiol groups and for forming a covalent bond
between the activated thiol groups and nucleic acids containing an
appropriate acrylamide functional group. Such nucleic acids can be
synthesized by the purchaser or, alternatively, they can be
purchased separately from the kit of the invention. Kits containing
components in addition to a solid support containing immobilized
thiol groups are also within the scope of the invention. Such kits
can contain components for activating the thiol groups, e.g.
reducing agents and/or a wash buffer. Such kits can also contain
nucleic acids with acrylamide functional groups. The nucleic acids
can be identical, non-identical or a combination can be provided.
Typically, components of the kits are contained in separate
containers.
[0154] In an alternate embodiment, a kit can contain a solid
support containing a plurality of latent thiol groups and nucleic
acids containing an appropriate acrylamide functional group. Kits
containing components in addition to a solid support containing
immobilized thiol groups and nucleic acids containing an
appropriate acrylamide functional group are also within the scope
of the invention. Such kits can contain components for activating
the thiol groups, e.g., reducing agents and/or a wash buffer.
Typically, components of the kits are contained in separate
containers.
[0155] The features and other details of the invention will now be
more particularly described and pointed out in the examples. It
will be understood that the particular embodiments of the invention
are shown by way of illustration and not as limitations of the
invention. The principle features of this invention can be employed
in various embodiments without departing from the scope of the
invention.
EXAMPLES
Example 1
Derivatization of Polystyrene Microspheres with an
Acrylamide-Functional Nucleic Acid
[0156] FIG. 1 depicts schematically one method for covalently
bonding acrylamide functional nucleic acids to a polystyrene
support. In Step 1, the formation of latent thiol groups on
amino-functional polystyrene is illustrated. Approximately 10 .mu.L
of amino-functional polystyrene microspheres (10% suspension) were
dispersed in 80 .mu.l of phosphate buffer (50 mM, pH=7.5). The
amino-functional polystyrene microspheres had a diameter of
approximately 1 .mu.m and an amino group density of approximately
75 .mu.eq/g (Bang's Laboratories Inc., Fisher, Ind.). To the
polystyrene microsphere suspension, 368 .mu.g of N-succinimidyl
S-acetylthioipropionate (hereinafter "SATP"), (Pierce, Rockford,
Ill.) in 10 .mu.l of dimethyl sulfoxide (hereinafter "DMSO") was
slowly added. This mixture was gently shaken for approximately two
(2) hours at ambient temperature. The microspheres were then washed
three (3) times, each time with 100 .mu.l of phosphate buffer (50
mM, pH=7.5), by adding the phosphate buffer and mixing,
centrifuging, and decanting the supernatant, to provide latent
thiol microspheres after the final decanting step.
[0157] (The latent thiol derivatized microspheres, also referred to
as thiolated microspheres, can optionally be dried and stored at
this point for future use. If dried, the thiolated microspheres can
be rehydrated in phosphate buffer prior to continuing with Step 2.)
The following steps were performed to provide oligonucleotide
functional polystyrene microspheres.
[0158] As illustrated, in Step 2, the activation of latent thiol
(deacetylation) was described. Deacetylation buffer was prepared
containing 50 mM phosphate buffer, 25 .mu.M EDTA and 0.5 M of
hydroxylamine HCl. It had a final pH of 7.5. Next, 100 .mu.l of the
deacetylation buffer was added to the latent thiol microspheres in
the centrifuge tube from Step 1. The centrifuge tube was gently
shaken for two (2) hours at ambient temperature. After
centrifugation, the supernatant and microspheres were separated by
decantation providing activated microspheres in the tube.
[0159] Step 3 describes oligonucleotide attachment. To the
centrifuge tube from step 2, 100 .mu.l of 1X TE buffer were added,
along with 1.0 .mu.l of acrylamide-modified oligonuleotide primer
pair solution having a concentration of 100 .mu.M for each
oligonucleotide (Operon Technologies, Alameda, Calif.). The
microsphere suspension was then gently shaken for one (1) hour at
ambient temperature. Oligonucleotides covalently bound through a
thioether linkage to activated microspheres (oliogonucleotide bound
microspheres) were obtained.
[0160] In Step 4, an optional step of blocking the excess reactive
thiol groups is described. Excess thiol groups on the
oligonucleotide bound microspheres can be blocked, if desired. To
block the excess thiol groups on the microspheres, 277 .mu.g of
iodoacetamide (Aldrich Chemical Co., Milwaukee, Wis.) dissolved in
10 .mu.l of 1X 10 mM Tris-HCL pH 8.3; 1 mM EDTA buffer (hereinafter
"TE buffer") was added to the oligonucleotide bound microspheres of
Step 3. The centrifuge tube and its contents were then shaken for
one (1) hour at ambient temperature. The TE buffer was decanted off
the microspheres. Then, the microspheres were washed three (3)
times with 100 .mu.l each of TE buffer to provide capped,
oligonucleotide bound microspheres. After decanting the last wash,
the capped oligonucleotide bound microspheres in the tube were
ready for use in a PCR reaction, for example, as illustrated in
U.S. Pat. No. 4,683,202, the disclosure of which is incorporated
herein by reference. (Alternatively, the capped oligonucleotide
microspheres can be dried and stored at this point for future use.
If dried, the capped oligonucleotide microspheres can be rehydrated
in phosphate buffer prior to use.)
Example 2
Array Formation on an Aminoalkyl Glass Slide
[0161] A glass slide having a plurality of amine groups attached in
a substantially uniform spatial pattern to a flat surface thereof
(Part #S 4651, aminoalkyl silane coated slides, Sigma Chemical Co.,
St. Louis, Mo., 1999 catalog) was submerged for two (2) hours at
ambient temperature in a solution of 15 mM SATP in 50 mM phosphate
buffer pH 7.5, 10% DMSO. The glass slide was then washed three (3)
times with 50 mM, pH 7.5 phosphate buffer by submerging the glass
slide in phosphate buffer. A glass slide having a plurality of
latent thiolated groups was formed.
[0162] The glass slide was submerged in a solution of deacetylation
buffer which contains 50 mM of phosphate buffer pH 7.5, 25 .mu.M of
EDTA and 0.5 M of hydroxylamine-HCl, for two (2) hours at ambient
temperature to provide a glass slide having a plurality of
activated thiol groups.
[0163] A plurality of acrylamide-modified oligonucleotides were
attached to the activated thiol groups. A lass slide was uniformly
modified with acrylamide-modified nucleic acids by submerging the
activated glass slide in a 100 .mu.M solution of
acrylamide-modified oligonucleotide in 1X TE buffer (Acrydite.TM.
acrylamide-modified oligonucleotide obtained from Operon
Technologies, Alameda, Calif.) for one (1) hour at ambient
temperature.
[0164] A plurality of acrylamide-modified oligonucleotides, each
having a different sequence, were deposited onto the slide in
spatially distinct regions. Deposition of the oligonucleotides onto
the activated array was performed manually, although it could also
have been performed automatically (e.g., using a pipetting robot).
Using a micropipette dipped into an acrylamide-modified
oligonucleotide solution, an aliquot of the solution was
transferred to a predetermined region on a lass slide having
activated thiol groups. A second acrylamide-modified olionucleotide
was then deposited onto a second region that is spatially distinct
from the first region, using the same procedure with a fresh
micropipette.
[0165] Alternatively, a capillary dispenser, for example, one as
illustrated in U.S. Pat. No. 5,807,522, the teachings of which are
incorporated herein by reference, can be used. Other spotting
methods known to those skilled in the art, which permit the regions
of the array to be arranged so that the oligonucleotide sequences
are appropriately spaced, can also be used.
[0166] In an alternative embodiment, random arrays are formed using
an ink-jet spray apparatus such as, for example, the apparatus
illustrated in U.S. Pat. No. 5,599,695, the teachings of which are
incorporated by reference in their entirety. In yet another
embodiment, regions of an array can be defined utilizing a mask,
such as those utilized in photolithography.
[0167] After deposition of all oligonucleotides on the array,
activated thiol groups that have not been covalently linked to an
acrylamide-modified oligonucleotides are blocked. For example, the
method of Step 4 in Example 1 above can be used to inactivate
remaining thiol groups. Other chemical treatments known to those of
skill in the art can also be utilized.
Example 3
Array Formation on a Polystyrene Support
[0168] A polystyrene flat support having a plurality of amine
groups attached in a substantially uniform spatial pattern to a
flat surface thereof is submerged for two (2) hours at ambient
temperature in a solution of 15 mM SATP in dimethyl
sulfoxide-phosphate buffer. Then, the polystyrene flat support is
washed three (3) times with 50 mM, pH=7.5 phosphate buffer,
submerging the polystyrene flat support in phosphate buffer to
provide a polystyrene flat support having a plurality of latent
thiolated sites.
[0169] Deacetylation buffer which contains 25 .mu.M EDTA, 0.5 M
hydroxylamine-HCl in 50 mM phosphate buffer is prepared with a
final pH of 7.5. It is mixed with 100 mM acrylamide-modified
oligonucleotides (Operon Technologies, Alameda, Calif.) in 1X TE
buffer. The solution is selectively spotted onto the latent thiol
sites in predefined regions. Since only selected areas on the
support are provided with activated thiol groups through contact
with the deacetylation buffer, only those regions are available for
binding acrylamide-modified oligonucleotides. Thus, latent regions
remain and can be used to separate the regions to which
oligonucleode has been covalently bound.
Example 4
Array Formation With Predefined Patterns
[0170] In yet another alternative embodiment, a glass slide can be
provided with amine groups in a predefined pattern. The amine
groups can then be converted to latent thiol groups and the support
treated as described in Example 2.
Example 5
Array Formation on an Acrylate Slide
[0171] This example demonstrates attachment of acrylamide-modified
oligonucleotide probes to a crosslinked polyacrylamide gel support
containing the disulfide bisacrylamide crosslinker
N,N'-bis(acryloyl)cystamine, (hereinafter "BAC", Fluka, Buchs,
Switzerland). Acrylamide groups on the oligonucleotide probes were
added during synthesis using commercially available acrylamide
phosphoramidites (Acrydite.TM. phosphoramidites I and III, Mosaic
Technologies, Waltham, Mass.). Solid phase hybridization
performance of 5'-methacrylamide-modifi- ed oligonucleotide probes,
indicated by "Ac1" (generated with Acrydite.TM. I) 220 were
compared with 5'-acrylamide-modified oliganucleotide probes,
indicated by "Ac3" (generated with Acrydite.TM. III) 210. In all
cases, (FIGS. 2A, 2B), oligonucleotide probes were spotted onto
thiol containing gel-coated slides and allowed to react. The slides
were washed to remove unbound probe, and then hybridized to a
fluorescently labeled oligonucleotide target to reveal the
hybridization performance of the immobilized probes. Control
experiments (not shown) demonstrated that when tris(2-carboxyethyl)
phosphine hydrochloride (hereinafter "TCEP") treatment was omitted,
images similar to FIG. 2B were produced, thus, demonstrating that
activated, reduced thiols (but not latent thiols) were required for
probe binding. FIG. 2B shows that 5'-acrylamide probe binding was
prevented by pretreating the TCEP-treated slide with excess monomer
acrylamide, suggesting that the acrylamide function of the probes
are important for binding. FIG. 2A also demonstrates that 5' amino
230 and 5'hydroxyl 240 modified oligonucleotide probes show low
binding to gels containing activated thiol groups.
[0172] Step 1: Preparation of Acrylate Slide Bound to Acrylamide
Gel Layer
[0173] Aqueous acrylamide solution was prepared using 6% acrylamide
(29:1 ratio of acrylamide monomer to bisacrylamide (BioRad
laboratories, Inc.; Hercules, Calif.) and 0.5% (wt/v)
N,N'-bis(acryloyl)cystamine (Fluka, Buchs, Switzerland)) in 100 mM
sodium borate buffer pH 9 hereinafter "SBB"). The aqueous solution
was cooled on ice. A 100 .mu.l aliquot was mixed with 1 .mu.l fresh
10% ammonium persulfate (hereinafter "APS", BioRad Laboratories,
Inc., Hercules, Calif.) and 1 .mu.l
N,N,N',N'-tetramethyl-ethylenediamine (hereinafter "TEMED"; BioRad,
Hercules, Calif.) diluted 10:1 with water to provide an acrylamide
gel solution. Next, 30 .mu.l of the acrylamide gel solution were
pipetted onto an acrylate slide (Cat# ACR-25C, CEL Associates,
Inc., Houston, Tex.) that is at room temperature. The acrylamide
gel solution was overlaid with a glass coverslip (24.times.50 mm)
taking care not to create any air bubbles or gaps. The acrylamide
gel solution was allowed to polymerize on the acrylate slide for at
least 45 minutes at room temperature. The coverslip was removed
leaving an acrylamide gel layer having latent thiol groups bound to
the acrylate slide.
[0174] (At this point, the slides can be also be dried and stored
for later use after rehydration.)
[0175] Step 2: Activation of the Latent Thio Groups
[0176] The acrylate slides each having a thio-derivatized
acrylamide gel layer were placed in 20 mM TCEP (Fluka; Buchs,
Switzerland) in 100 mM SBB pH 9 and were incubated for 15
minutes.
[0177] The slides were washed two (2) times in TE buffer, then
rinsed with water and allowed to air dry.
[0178] Step 3: Oligonucleotide Attachment to Acrylamide Gel
[0179] The slides were spotted within 30 minutes of TCEP treatment
with oligonucleotides modified as described. Spotting solutions
were prepared with 100 mM SBB pH 9 and 20 .mu.M oligonucleotide
(Operon, Alameda, Calif.) containing 5' Acrydite III modification,
5' Acrydite I modification, 5' NH.sub.2 modification, or without a
5' modification. Individual spots of 0.5 .mu.l of each solution (10
pmoles) were placed onto each slide in triplicate. The slides were
placed in a nitrogen box and incubated for one (1) hour at room
temperature. Then, the slides were washed two (2) times with
TE+0.2M sodium chloride (hereinafter "NaCl"). The slides were
washed two (2) times in TE pH8 and allowed to dry.
[0180] Step 4: Oligonucleotide Detection By Hybridization
[0181] An aliquot of 60 .mu.l hybridization mix (10 .mu.M
complementary fluorescent oligonucleotide (OPERON, Alameda, Calif.)
in 5X SSPE+0.2% SDS was placed on the slide and the slide was
overlaid with a coverslip. The slides were allowed to hybridize for
one (1) hour at room temperature in a humid hybridization chamber
(Corning, Corning, N.Y.). Amer one (1) hour, the slides were washed
two (2) times with 1X SSPE+0.1%, SDS. Then, each slide was washed
one (1) time with TE at pH 8 and was allowed to air dry.
[0182] The slide is imaged dry in a fluorescent imager (Molecular
Dynamics, Fluorimager 595, Sunnyvale, Calif.).
Example 6
Microarray Formation on an Gel-Coated Support and Comparison of
Blocking Agents
[0183] Step 1: Preparation of Gel-Coated Slide Supports
[0184] A polymerization solution was prepared with 6% acrylamide
(29:1), and 0.5% BAC (wt/v), in 100 mM SBB pH9. (BAC required
heating and vortexing to go into solution). 1 .mu.l fresh 10% APS
(made same day) and 1 .mu.l of 10:1 dilution of H.sub.2O:TEMED were
added to 100 .mu.l of the solution and mixed thoroughly. 10 .mu.l
of solution were pipetted onto an Acrylate Slide (CEL Associates,
Inc., ACR-25C) and overlaid with a lass overslip (18.times.18 mm),
taking care not to create any air bubbles or gaps in the solution.
The acrylamide layer was allowed to polymerize at least 20 minutes
at room temperature. After the slides were rinsed in TE and allowed
to air dry, they were ready for spotting of oligo.
[0185] Step 2: Activation of the Latent Thiol Group
[0186] Spotting solutions were prepared from 20 .mu.M of
Acrydite.TM. oligo and 100 mM TCEP, all in 100 mM SBB at pH 9.
[0187] 35 .mu.l of various solutions were prepared and placed in a
microtiter plate.
1TABLE 1 Well Well Well Pin Slide Slide # oligo Col Row Probe
Replicate # Abs Abs A1 Tryp 370 1 1 0 1 5.5 40 A3 Tryp 355 3 1 0 1
5.8 40 A5 Tryp 575 5 1 0 1 6.1 40 A7 no oligo 7 1 0 1 6.4 40 A9
blank 9 1 0 1 6.7 40 A11 blank 11 1 0 1 7 40 A2 Bglobar1269 2 1 0 2
14.5 40 A4 Bglobar1287 4 1 0 2 14.8 40 A6 Bglobar490 6 1 0 2 15.1
40 A8 ANF401 8 1 0 2 15.4 40 A10 no oligo 10 1 0 2 15.7 40 A12
blank 12 1 0 2 16 40
[0188] Step 3: Oligonucleotide Attachment to Acrylamide Gel
[0189] The slides were arrayed on a GMS spotter as follows:
[0190] The slides were incubated on a lab bench at room temperature
for one (1) hour. After one (1) hour, the slides were soaked for 30
minutes in 20% dimethylacrylamide (hereinafter "DMA") or 20%
2-hydroxyethylmethacrylate (hereinafter "HEMA") in 100 mM SBB at pH
9. The slides were washed two (2) times with TE+0.2M NaCl. Then,
the slides were washed once in TE and allowed to dry.
[0191] Step 4: Oligonucleotide Detection by Hybridization
[0192] Adhesive hybridization chambers were attached to the slides
and 90 .mu.l of the hybridization mixture were added to slides:
cDNA prepared from 50 ng input globin RNA in 4X SSPE+0.2% Tween.
The slides were hybridized overnight at 55.degree. C. in a humid
hybridization chamber. After incubation, the slides were washed two
(2) times with 1XSSPE+0.1% Tween. Then, the slides were washed one
(1) time with TE and allowed to air dry. The slides were imaged
dry.
[0193] When quantified, blocking with HEMA is comparable to slide
with DMA. Background with HEMA block is slightly higher, but the
difference is not significant. The results, both when blocked with
DMA and blocked with HEMA, are shown in FIGS. 3A-3B.
Example 7
Preparation of 0.5% bis(acryloyl) Cystamine (BAC) Thin Gel
Supports
[0194] The following were mixed in a 15 ml tube:
2 Final conc: 0.5 ml dimethylformamide (DMF) 5.0% 50 mg
N,N'-bis(acryloyl)cystamine (BAC) 0.5% (19.2 mM) 1.5 ml 40% stock
acrylamide/bis solution 6.0% (844 mM) 2.0 ml 500 mM Tris-Glycine
buffer pH 9.0 100 mM 6.0 ml water 10 ml total volume
[0195] 1 ml of the above solution was placed on ice and added
to:
3 1 .mu.l 1% SDS 0.001% 10 .mu.l 10% aqueous APS 0.1% 10 .mu.l 10%
aqueous TEMED 0.1%
[0196] 10 .mu.of the above solution was pippetted onto a microscope
that was coated with an acrylic silane (CEL Associates, Inc.,
Houston, Tex.) and overlaid with a lass coverslip (18.times.18 mm)
taking care not to create any air bubbles or gaps in the solution.
The solution was allowed to polymerize for 30 minutes at room
temperature. The coverslip was removed using a razor blade. The
slides were washed in TE buffer and allowed to dry at room
temperature.
Example 8
Preparation of
4[(1-oxo-3-[[2-[(1-oxo-2-propenyl)-amino]ethyl]dithio]propy-
l]amino butanoic acid (AEMA) Thin Gel Supports
[0197] 123 mg of AEMA were dissolved in 0.5 ml dimethyl formamide
(hereinafter "DMF") and 1.5 ml water. After the AEMA was dissolved
the following was added:
4 Final conc: 1.5 ml 40% stock acrylamide/bis solution 6.0% (844
mM) 2.0 ml 500 mM Tris-Glycine buffer pH 9.0 100 mM 5.0 ml water 10
ml total
[0198] 1 ml of the above solution was taken, placed on ice and the
following was added:
5 1 .mu.l 1% SDS 0.001% 10 .mu.l 10% aqueous APS 0.1% 10 .mu.l 10%
aqueous TEMED 0.1%
[0199] 10 .mu.l of the above solution was pippetted onto a
microscope that was coated with an acrylic silane (CEL Associates,
Inc. Houston, Tex.) and overlaid with a glass coverslip
(18.times.18 mm) taking care not to create any air bubbles or gaps
in the solution. The solution was allowed to polymerize for 30
minutes at room temperature. The coverslip was taken off using a
razor blade. The slides were washed in TE buffer and allowed to dry
at room temperature.
Example 9
Preparation of Thin Gel Supports with Reduced BAC,
.beta.-Mercaptoethanol Method
[0200] The following was added to a 15 ml tube:
6 100 mg BAC 1.0% (0.384 mmole) 0.5 ml DMF 0.5 ml water
[0201] After BAC dissolved, the following was added:
7 27.5 ml .beta.-mercaptoethanol dissolved in 0.5 ml water (0.384
mmole)
[0202] The solution was allowed to react for 1-12 hours at room
temperature. After incubation the following was added:
8 Final conc: 1.5 ml 40% stock acrylamide/bis solution 6.0% (844
mM) 2.0 ml 500 mM Tris-Glycine buffer pH 9.0 100 mM 5.0 ml water 10
ml total
[0203] 1 ml of the above solution was taken, placee on ice and the
following was added:
9 1 .mu.l 1% SDS 0.001% 10 .mu.l 10% aqueous APS 0.1% 10 .mu.l 10%
aqueous TEMED 0.1%
[0204] 10 .mu.l of the above solution was pippetted onto a
microscope that was coated with an acrylic silane (CEL Associates,
Inc. Houston, Tex.) and overlaid with a glass coverslip
(18.times.18 mm) taking care not to create any air bubbles or gaps
in the solution. The solution was allowed to polymerize for 30
minutes at room temperature. The coverslip was taken off using a
razor blade. The slides were washed in TE buffer and allowed to dry
at room temperature.
Example 10
Preparation of Thin Gel Supports with Reduced BAC, Thioacetic Acid
Method
[0205] The following was added to a 15 ml tube:
10 100 mg BAC 1.0% 0.384 mmole 0.5 ml DMF 0.5 ml water
[0206] After the BAC was dissolved, the following was added:
11 43.8 mg thioacetic acid 0.384 mmole
[0207] After the incubation, the following was added:
12 Final conc: 1.5 ml 40% stock acrylamide/bis solution 6.0% (844
mM) 2.0 ml 500 mM Tris-Glycine buffer pH 9.0 100 mM 5.0 ml water 10
ml total
[0208] 1 ml of the above solution was taken, placed on ice and the
following was added:
13 1 .mu.l 1% SDS 0.001% 10 .mu.l 10% aqueous APS 0.1% 10 .mu.l 10%
aqueous TEMED 0.1%
[0209] 10 .mu.l of the above solution was pippetted onto a
microscope that was coated with an acrylic silane (CEL Associates,
Inc. Houston, Tex.) and overlaid with a glass coverslip
(18.times.18 mm) taking care not to create any air bubbles or gaps
in the solution. The solution was allowed to polymerize for 30 min.
at room temperature. Take off the overslip using a razor blade.
Wash the slides in TE buffer and allow to dry at room
temperature.
Example 11
Comparison of BAC, AEMA, and Reduced BAC Supports for Microarray
Hybridization; Effect of Buffer and Glycerol in Spotting
Solutions
[0210] Three different types of supports were prepared.
[0211] Standard support containing 0.5% BAC (19.2 mM disulfide
bonds yielding 38.4 mM thiol groups after reduction). The
preparation is described in Example 7 above.
[0212] AEMA support containing 38.4 mM AEMA and 38.4 mM thiol
groups bound to the gel after reduction. The preparation is
described in Example 8 above.
[0213] BAC+ME gel pad containing 38.4 mM BAC and 38.4 mM thiol
groups bound to the gel after reduction. The preparation is
described in Example 9 above.
[0214] After polymerization, two slides of each type were washed
and treated with 10 mM TCEP solution in 100 mM sodium carbonate, pH
10 for 20 min. Another slide prepared with a gel layer containing
no BAC was treated the same way. The slides were washed four (4)
times in 1X SSPE buffer containing 0.1% SDS. Then the slides were
washed two (2) times in 10 mM TE buffer pH 8.
[0215] The conversion of disulfide groups into thiol groups was
confirmed by spotting 0.5 .mu.l of 1 mM
5,5'-dithio-bis-(2-nitrobenzoic acid) (hereinafter "DTNB") solution
in 100 mM phosphate buffer pH 8 on the gel. The spot turned yellow
on AEMA and BAC containing gel layers, but remained colorless on a
control slide with just acrylamide gel layer. This indicated that
all TCEP was eluted from gel layers.
[0216] A series of solutions of 50 .mu.l volume was prepared for
spotting on slides. The solutions contained different
concentrations (3, 10 and 30 mM) of Acrydite.TM.-modified DNA
olionucleotide BD 1216 (complementary probe for rabbit globin cDNA
target) in either 100 mM Tris-Glycine pH 9 or 100 mM sodium
carbonate pH 10 buffer. Also, solutions containing 10 mM oligo and
10% or 20% glycerol were prepared. (Glycerol containing solutions
are less sensitive to humidity of air during spotting and give
higher yields of DNA probes binding in low and moderate humidity)
In this experiment, all gel solutions were reduced with TCEP before
spotting and no TCEP was added into spotting solutions.
[0217] The solutions were placed in a microplate well (Microseal 96
V-bottom microplates, MJ Research, Mass.) and the arrays were
spotted using a Genetic Microsystems 417 Arrayer (Affymetrix, Santa
Clara, Calif.) and incubated overnight at room temperature.
[0218] The residual activated thiols were quenched by soaking the
slides in 10% acrylamide solution, 100 mM sodium carbonate buffer
pH 10, for 20 minutes. to improve background between spots, then
washed two (2) times in 10 mM TE+200 mM NaCl, followed by two (2)
times in 10 min TE and dried.
[0219] Hybridization was carried out overnight at 55.degree. C. in
plastic chambers with rabbit globin cDNA labeled with Cy3
fluorescent dye. Concentration of cDNA was 50 ng/ml of
hybridization buffer (4X SSPE containing 0.02% Tween 20). After
hybridization, the slides were washed three (3) times in 1X SSPE
buffer and briefly washed two (2) times in 10 mM TE buffer, then
dried using nitrogen.
[0220] The arrays were scanned with ScanArray 4000 scanner (GSI
Lumonics, Watertown, Mass.) using green line 543.5 nm of HeNe laser
for excitation. The laser power was set at 90% and photomultiplier
power (PMT) was set at 60%. The data was analyzed using ImageQuant
5.1 software (Molecular Dynamics, Sunnyvale, Calif.). The
background signal from an unspotted position on the microarray was
subtracted from the total fluorescence signal of each hybridized
probe spot.
[0221] The corrected fluorescence intensity data are plotted in
FIGS. 6 and 7. FIG. 7 compares the Tris-Glycine buffer with the
carbonate buffer. At each concentration of probe oligonucleotide
tested, better hybridization signals were obtained when the probes
were spotted with the carbonate buffer system. FIG. 7 shows data
only for the 10 .mu.M probe spots.
[0222] In addition, slightly better signals were obtained using the
monofunctional disulfide acrylamide, AEMA. Similar enhancement of
hybridization signals resulted from using a gel layer containing
BAC that had been reduced with an equimolar amount of
mercaptoethanol prior to gel polymerization. Similar hybridization
enhancements were also obtained from gel layers containing BAC that
had been reduced with thioacetic acid prior to gel formation, as
described in Example 10 (data not shown). While not wishing to be
bound by theory, the enhancement may result from the fact that BAC
derived thiols in gels cast with unreduced BAC, as in the BAC
protocol of Example 7, may be held in close proximity after
reduction in the gel, and therefore may reform the disulfide,
thereby reducing the number of thiol groups available for probe
binding.
Example 12
Preparation of Acrylate Slides with 1-6% BAC Polymerization in
Organic Solvent without Comonomer
[0223] Acrylate slides were co-polymerized in BAC solutions
containing concentrations of BAC ranging from 1% to 6%. A 1% BAC
coated slide was made by mixing 3 ml of 10% BAC in DMF; 12 ml of
DMF; 15 ml of water and 600 .mu.L of 25% APS; 100 .mu.L TEMED.
After mixing, this solution was dispensed into a container with
four acrylate slides; the solution was allowed to polymerize
overnight at room temperature. A white homogenous gel-like material
signaled the visible onset of polymerization. The BAC acrylate
slides were then removed from the solution and rinsed in deionized
water with gentle rubbing to remove the visible white film formed
on the BAC acrylate slide.
[0224] After treatment with TCEP to generate active thiols, the
slides were spotted with an Acrydite.TM. oligonucleotide (50mer)
designed to hybridize to cDNA transcribed from the mRNA of the
rabbit beta-globin gene. The concentrations of 30-mer used for the
spots were 30 .mu.M, 10 .mu.M, 5 .mu.M, 1 .mu.M, and 0 .mu.M.
Acrydite.TM. modified oligonucleotide bound to the BAC acrylate
slide was hybridized overnight at 55.degree. C. in 4X SSPE; 0.02%
Tween20.RTM. to Cy3-dUTP labeled cDNA (prepared from rabbit
reticulocyte polyA+mRNA (Gibco-BRL; Life Technologies; Rockville,
Md.) with an arrayTRACKER.TM. Standard Labeling cDNA Kit (Cat.
#490-100, Displays Systems Biotech, Inc.; Vista, Calif.) in
accordance with the instructions provided with the kit using the
following modification: After the final precipitation in the
display systems protocol, the cDNA preparation was resuspended in
40 .mu.L buffer (4X SSPE; 0.02% Tween20.RTM.), and this mixture was
run through a G25 spin column (Cat. # 27-5325-01 Amersham
Pharmacia, Microspin G-25 column). The hybridized spotted slide was
washed three (3) times in 1X SSPE buffer containing 0.02%
Tween20.RTM., then in TE, and then dried with a stream of nitrogen.
The hybridized oligonucleotide spotted slide was imaged with a GSI
Lumonics ScanArray 4000 Microarray Analysis System (GSI Lumonics,
Inc.; Billerica, Mass.).
[0225] The results demonstrated that the amount of cDNA bound
depends on the amount of BAC used to prepare the slide. Optimal
signals are seen with BAC concentrations of 1-3%
14TABLE 2 Effect of BAC conc slide % BAC Signal Background 01 1
3,839 71 05 0.5 1,798 52 06 1.5 4,275 67 07 3 7,479 75 08 6 3,315
83 Signal-sum of the RFUs (in thousands) for each pixel in an area
corresponding to the region of the spotted oligo Back-sum of the
RFUs (in thousands) for each pixel in the area of equal size, where
no oligo was spotted.
Example 13
Preparation of Acrylate Slides with BAC-Comonomer-Polymerization in
Organic Solvent
[0226] A procedure similar to that in Example 7 was used to make
slides with 2% BAC, with various amounts of P400 mm. To make a
slide with a coating of 2% BAC-1% P400 mm, four acrylate slides
were immersed in a solution made by mixing: 3.6 ml of 10% BAC in
DNEF, 5.4 ml of DMF; 9 ml of water; 180 .mu.L of P400 mm; 240 .mu.L
of 25% APS; 40 .mu.L TEMED After standing at room temperature, the
appearance of the solution was noted. The BAC acrylate slides were
then removed from the solution and rinsed in deionized water. In
cases where a film was visible on the slides, gentle rubbing was
used to remove the visible white film. The BAC acrylate slides were
again washed in water, and then dried with a stream of
nitrogen.
[0227] After treatment with TCEP to generate active thiols, the
slides were spotted with an Acrydite.TM. modified oligonucleotide
(50mer) designed to hybridize to cDNA transcribed from the mRNA of
the rabbit beta-globin gene. The concentrations of 50-mer used for
the spots were 30 .mu.M, 10 .mu.M, 5 .mu.M, 1 .mu.M, and 0 .mu.M.
Acrydite.TM. modified oligonucleotide bound to the BAC acrylate
slide was hybridized overnight at 55.degree. C. in 4X SSPE; 0.02%
Tween20.RTM. to Cy3-dUTP labeled cDNA (prepared from rabbit
reticulocyte polyA+mRNA (Gibco-BRL; Life Technologies, Rockville,
Md.) with an arrayTRACKER.TM. Standard Labeling cDNA Kit, (Cat.
#490-100, Displays Systems Biotech, Inc.; Vista, Calif.) in
accordance with the instructions provided with the kit, except that
after the final precipitation in the display systems protocol, the
cDNA preparation was resuspended in 40 .mu.L of buffer (4X SSPE;
0.02% Tween20.RTM.), and this mixture was run through a G25 spin
column (Cat. #27-5325-01, Amersham Pharmacia, Microspin G-25
column). The hybridized spotted slide was washed three (3) times in
1X SSPE buffer containing 0.02% Tween20.RTM., then in TE, and then
dried with a stream of nitrogen. The hybridized oligonucleotide
spotted slide was imaged with a GSI Lumonics ScanArrayR 4000
Microarray Analysis System (GSI Lumonics, Inc.; Billerica,
Mass.).
[0228] Addition of P400 mm in the range of 0.5 to 4% (v/v) was seen
to change the nature of the precipitate formed during
polymerization reaction. Depending on the concentration of P400 mm,
the solution formed a clear gel, a cloudy gel, or no visible gel
i.e., remained a liquid. When the solution remained a liquid, no
film was formed on the slides, and the rubbing step above was not
necessary.
[0229] The following Table shows the results obtained for slides
prepared with different concentrations of P400 mm in 2% BAC.
15 TABLE 3 Back- Slide % Signal* ground* Film Appearance # P400 mm
Rfu Rfu After Polymerization 01 0.0 796 97 White, soft gel 02 0.5
3,188 26 White, soft gel 03 1.0 4,243 68 Bluish, grey firm gel 04
2.0 2,530 45 Clear, liquid 05 4.0 637 34 Clear, firm gel *Signal is
the sum of the RFUs (in thousands) for each pixel in an area
corresponding to the region of the spotted oligonucleotide.
**Background is the sum of the RFUs (in thousands) for each pixel
in the area of equal size, where no oligonucleotide was
spotted.
[0230] The data also showed that the amount of cDNA bound was
dependent on both the amount of comonomer, and the concentration of
50-mer oligo used in spotting, as shown in the following table:
16TABLE 4 Effect of Conc of spotted oligo slide oligo % BAC % p400
mm signal S-B S/B 01 30 2 0 796 699 8.2 10 2 0 307 210 3.2 5 2 0
245 148 2.5 1 2 0 150 53 1.5 0 2 0 97 0 1.0 03 30 2 1 4,243 4,175
62 10 2 1 2,200 2,132 32 5 2 1 1,308 1,240 19 1 2 1 406 338 5.9 0 2
1 68 0 1.0 Oligo-concentration (micromolar) of oligo spotted onto
thiol slide. Slide 01 was prepared with 2% BAC Slide 05 was
prepared with 2% BAC/1% p400 mm
[0231] Signal--sum of the RFUs for each pixel in an area
corresponding to the region of the spotted oligo
[0232] S-B signal minu the signal for spot with 0 oligo.
[0233] S/N singal divided by the signal for spot with 0 oligos.
[0234] Addition of comonomer also results in a change in the size
of the spot made by the oligonucletide in solution. FIG. 4 shows a
plot of fluorescent intensity across a spot for slides prepared
with 2% BAC or 2% BAC plus P400 mm at the different
concentrations.
Example 14
Preparation of Acrylate Slides with BAC-Polymerization in Water
[0235] 1% BAC in water coated slides were prepared as follows: 0.5
g of BAC was dissolved in 50 ml of deionized water at 70.degree. C.
Acrylate slides were completely submerged in the heated BAC
solution. 1.0 ml of 0.05% APS and 1.0 ml of 0.05% TEMED were added
The container was sealed and shaken for 1 minute. The
polymerization reaction was complete within several minutes. A
white precipitate of polyBAC formed. After removal of the
macroscopic polyBAC particulate with water, the acrylate slides
appeared coated with a homogeneous thin white film. This film was
removed by gentle scrubbing under water. The resulting dried BAC
acrylate slides appeared clear and transparent with no visible
residue.
[0236] A BAC acrylate slide was then spotted with different
concentrations ranging from 30 .mu.M to 1 .mu.M of
beta-globin-specific 70mer Acrydite.TM. modified oligonucleotide
containing TCEP in the spotting solution. The spots were visualized
by hybridization with Cy3 labeled globin cDNA (10 ng/80 ul) in 100
.mu.l of 20.times. saline sodium phosphate EDTA buffer (SSPE; 3.6 M
sodium chloride, 200 mM sodium phosphate, pH 7.4, 20 mM EDTA, pH
7.4) in a hybridization chamber. The visualized spots formed are
shown in FIG. 5, a photograph of the BAC acrylate slide after
hybridization to a fluorescent complementary oligonucleotide
probe.
[0237] When the dried BAC acrylate slide were soaked in 50 mM TCEP
for 30 minutes prior to spotting and TCEP was left out of the
spotting solution, the results shown in FIG. 5B were obtained. When
SDS at 0.01% in carbonate buffer (100 MM, pH 10.0) was used as the
spotting buffer with the Acrydite.TM. modified oliogonucleotide on
a dried BAC acrylate slide exposed to TCEP for 30 minutes prior to
spotting, the results shown in FIG. 3C were obtained.
Example 15
Array Formation on a Mesh
[0238] A piece of nylon screen is placed between two silane treated
glass plates. An edge of the nylon screen is allowed to extend from
between the plates. A measured aliquot of the gel solution from
Example 5 is placed on the extension and the solution is wicked
onto the nylon screen between the two glass plates. The solution is
allowed to gel. Prior to use the slide is activated and provided
with Acrydite.TM. modified oligonucleotides as above.
Example 16
Use of Gel Matrix Coated Support
[0239] Provide a polyacrylamide gel matrix wherein the
polyacrylamide matrix has dithiol cross-linkages (no nucleic acid
or protein probes) such as, for example, by following Example 5,
Step 1. In addition to the reagents for forming the
thiol-derivatized acrylamide gel solution, mix in a desired cell
type for culture. For example, E. coli provided with nutrient
culture reagents may be grown within the polymerized gel. To
release the bacterial cells after replication has occurred, cleave
the dithiols to the degree desired, for example by following the
procedure described in Example 5, step 2 to release the cells.
[0240] Varying the amount of acrylamide cross linker used will
allow regulation of the density of the gel.
Example 17
Use of Gel Matrix Probe Bearing Coated Glass Slide Support for
Cloning and Amplification
[0241] Following the procedure described in Example 5, Step 1 using
a bisacrylamide cross-linker with bound Acrydite.TM. modified
oligonucleotides having a desired primer sequence to provide a
thiol derivatized acrylamide gel solution additionally having bound
oligonucleotides. To this gel solution, add the reagents (to
include a second primer in solution where desired; nucleic acids;
enzyme) required to allow amplication by polymerase chain reaction
cycling and a sample thought to contain the nucleic acid fragment
to be amplified. Allow the gel solution to polymerize on a support
such as the acrylate slide and expose the slide to PCR cycling
conditions. Cleave the latent thiol groups using a procedure such
as that described in Example 5, Step 2. Release and remove the
amplified nucleic acid.
Example 18
Thin Layer Monomers Containing Disulfide Linkages For
Immobilization of Nucleic Acids
[0242] Acryl-silane coated microscope slides (Gel Associates, Inc.,
Houston, Tex.) were submerged in 0.1-2% heated BAC/water solution.
100-400 .mu.l or 10% APS and 10% TEMED were added per 50 ml of
solution for a rapid polymerization. Chain terminators, e.g.,
isopropanol, can be added prior to polymerization to induce short
chain growth. N,N' methylenebisacrylamide (hereinafter "BIS") can
be added to the BAC solution at the appropriate concentration prior
to polymerization to allow multiple chain growth or polymer
branching. Polymerization is complete within 5 minutes or upon the
formation of a white particulate (polyBAC). The white particulate
can be removed from the slides under water by a gentle hand scrub.
Slides were suspended in 5-100 mM TCEP for reduction of disulfides.
Thiol formation was immediately evident by development of a putrid
odor. The slides were then dried for spotting.
[0243] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those of skill in the art that various changes in
form and details may be made herein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *