U.S. patent application number 14/445103 was filed with the patent office on 2015-01-15 for methods and compositions for selective labeling of different biotinylated targets within multicolor or multilabel assays.
The applicant listed for this patent is Affymetrix, Inc.. Invention is credited to Stephen P. A. FODOR, Robert G. KUIMELIS, Glenn H. MCGALL.
Application Number | 20150018253 14/445103 |
Document ID | / |
Family ID | 46753704 |
Filed Date | 2015-01-15 |
United States Patent
Application |
20150018253 |
Kind Code |
A1 |
KUIMELIS; Robert G. ; et
al. |
January 15, 2015 |
Methods and Compositions for Selective Labeling of Different
Biotinylated Targets within Multicolor or Multilabel Assays
Abstract
Disclosed are compositions and methods for the labeling of two
or more targets with different labels. Specifically, disclosed are
compositions for biotin and the protection of biotin within
multilabel assays which employ the biotin-biotin binding protein
binding relationship for each distinct label in relation to targets
such as nucleic acids, polypeptides, antibodies or cells. These
multilabel assays are enabled through the use of biotin with
desthiobiotin, orthogonal protecting schemes for biotin, or a
combination of the approaches.
Inventors: |
KUIMELIS; Robert G.; (Palo
Alto, CA) ; MCGALL; Glenn H.; (Palo Alto, CA)
; FODOR; Stephen P. A.; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Affymetrix, Inc. |
Santa Clara |
CA |
US |
|
|
Family ID: |
46753704 |
Appl. No.: |
14/445103 |
Filed: |
July 29, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13329175 |
Dec 16, 2011 |
8835125 |
|
|
14445103 |
|
|
|
|
61424301 |
Mar 2, 2011 |
|
|
|
Current U.S.
Class: |
506/26 ;
506/23 |
Current CPC
Class: |
Y10T 436/25375 20150115;
Y10T 436/105831 20150115; Y10T 436/25125 20150115; Y10T 436/10
20150115; C07K 1/13 20130101; G01N 33/582 20130101; Y10T 436/107497
20150115 |
Class at
Publication: |
506/26 ;
506/23 |
International
Class: |
C07K 1/13 20060101
C07K001/13; G01N 33/58 20060101 G01N033/58 |
Claims
1. A method of labeling a plurality of targets with a plurality of
labels, the method comprising: providing a plurality of targets,
wherein the plurality of targets comprises a first target and a
second target, wherein the first target includes a first biotin
molecule, and wherein the second target includes a second biotin
molecule; binding the first biotin molecule with a first biotin
binding protein, wherein the first biotin binding protein includes
a first label; and binding the second biotin molecule with a second
biotin binding protein, wherein the second biotin binding protein
includes a second label.
2. The method of claim 1, wherein the plurality of targets
comprises polynucleotides.
3. The method of claim 1, wherein the plurality of targets
comprises polypeptides.
4. The method of claim 1, wherein the plurality of targets
comprises antibodies.
5. The method of claim 1, wherein the plurality of targets
comprises cells.
6. The method of claim 1, wherein the plurality of targets was
biotinylated to incorporate the first biotin molecule and the
second biotin molecule.
7. The method of claim 1, wherein the plurality of targets
additionally comprises a third target and a fourth target, wherein
the third target includes a third biotin molecule, and wherein the
fourth target includes a fourth biotin molecule.
8. The method of claim 1, wherein the first biotin molecule and the
second biotin molecule comprise biotin molecules with distinct
binding affinities for biotin binding proteins.
9. The method of claim 8, wherein the first biotin molecule
comprises biotin and the second biotin molecule comprises
desthiobiotin.
10. The method of claim 1, wherein at least one of either the first
biotin binding protein or the second biotin binding protein is
streptavidin.
11. The method of claim 1, wherein at least one of either the first
biotin binding protein or the second biotin binding protein is a
recombinant protein.
12. The method of claim 1, wherein the first biotin binding protein
is a different protein than the second biotin binding protein.
13. The method of claim 1, wherein the first label is
distinguishable from the second label.
14. The method of claim 13, wherein the first label and the second
label are fluorophores, wherein the first label has a first
emission spectrum, wherein the second label has a second emission
spectrum, and wherein the first emission spectrum is at least
partially non-overlapping with the second emission spectrum.
15. The method of claim 1, wherein the second biotin molecule is
protected by a protecting group.
16. The method of claim 15, wherein the protecting group is a
photolabile protecting group.
17. The method of claim 15, wherein the protecting group is an
acid-labile protecting group.
18. The method of claim 15, wherein the protecting group is a
base-labile protecting group.
19. The method of claim 15, wherein the protecting group is
connected to the second biotin molecule through an ether
linkage.
20. The method of claim 19, wherein the protecting group is
6-nitropiperonyloxymethyl.
21. The method of claim 17, wherein the protecting group is
dimethoxytrityl.
22. The method of claim 15, wherein binding the second biotin
molecule with the second biotin binding protein comprises: removing
the protecting group from the second biotin molecule; and binding
the second biotin binding protein to the second biotin
molecule.
23. The method of claim 22, wherein the first biotin molecule is
protected by a second protecting group, and wherein binding the
first biotin molecule with the first biotin binding protein
comprises: removing the second protecting group from the first
biotin molecule; and binding the first biotin binding protein to
the first biotin molecule.
24. The method of claim 22, wherein the plurality of targets
additionally comprises a third target and a fourth target, wherein
the third target includes a third biotin molecule, wherein the
fourth target includes a fourth biotin molecule, wherein the third
biotin molecule is protected by a second protecting group, wherein
the fourth biotin molecule is protected by a third protecting
group, and wherein the protecting group of the second biotin
molecule, the second protecting group and the third protecting
group are each different protecting groups.
25. The method of claim 24, additionally comprising: binding the
third biotin molecule with a third biotin binding protein, wherein
the third biotin binding protein includes a third label, and
wherein the second protecting group is removed to allow the third
biotin molecule to bind with the third biotin binding protein; and
binding the fourth biotin molecule with a fourth biotin binding
protein, wherein the fourth biotin binding protein includes a
fourth label, and wherein the third protecting group is removed to
allow the fourth biotin molecule to bind with the fourth biotin
binding protein.
26. The method of claim 25, wherein the protecting group of the
second biotin molecule, the second protecting group and the third
protecting group are each removed through a distinct removal
mechanism.
27. The method of claim 26, wherein the distinct removal mechanism
includes removal of at least one type of protecting group through
radiation comprising one or more selected wavelengths.
28. The method of claim 26, wherein the distinct removal mechanism
includes removal of at least one type of protecting group through
an acid treatment.
29. The method of claim 28, wherein the acid treatment comprises
treatment with an acid solution containing an acid selected from
the group consisting of trifluoroacetic acid, acetic acid and
monopotassium phosphate.
30. The method of claim 26, wherein the distinct removal mechanism
includes removal of at least one type of protecting group through a
base treatment.
31. The method of claim 9, wherein binding the desthiobiotin with
the second biotin binding protein comprises: displacing the
desthiobiotin from the first biotin binding protein with a biotin
solution; and binding the desthiobiotin with the second biotin
binding protein.
32. The method of claim 31, wherein the plurality of targets
additionally comprises a third target and a fourth target, wherein
the third target includes a third biotin molecule protected by a
first protecting group, wherein the fourth target includes a fourth
biotin molecule protected by a second protecting group, and wherein
the first protecting group and the second protecting group are
different protecting groups.
33. The method of claim 32, additionally comprising: binding the
third biotin molecule with a third biotin binding protein, wherein
the third biotin binding protein includes a third label, and
wherein the first protecting group is removed to allow the third
biotin molecule to bind with the third biotin binding protein; and
binding the fourth biotin molecule with a fourth biotin binding
protein, wherein the fourth biotin binding protein includes a
fourth label, and wherein the second protecting group is removed to
allow the fourth biotin molecule to bind with the fourth biotin
binding protein.
34. The method of claim 32, wherein the first biotin molecule is
protected by a third protecting group, and wherein the second
biotin molecule is protected by a fourth protecting group.
35. The method of claim 33, wherein the first protecting group and
the second protecting group are each removed through a distinct
removal mechanism.
36. The method of claim 35, wherein the distinct removal mechanism
includes removal of at least one type of protecting group through
radiation comprising one or more selected wavelengths.
37. The method of claim 35, wherein the distinct removal mechanism
includes removal of at least one type of protecting group through
an acid treatment.
38. The method of claim 37, wherein the acid treatment comprises
treatment with an acid solution containing an acid selected from
the group consisting of trifluoroacetic acid, acetic acid and
monopotassium phosphate.
39. The method of claim 35, wherein the distinct removal mechanism
includes removal of at least one type of protecting group through a
base treatment.
40. A method of labeling a plurality of targets with a plurality of
labels, the method comprising: providing a plurality of targets,
wherein the plurality of targets comprises a first target, a second
target, a third target and a fourth target, wherein the first
target includes a first biotin molecule, wherein the second target
includes a second biotin molecule protected by a first protecting
group, wherein the third target includes a third biotin molecule
protected by a second protecting group, and wherein the fourth
target includes a fourth biotin molecule protected by a third
protecting group; binding the first biotin molecule with a first
biotin binding protein, wherein the first biotin binding protein
includes a first label; binding the second biotin molecule with a
second biotin binding protein, wherein the second biotin binding
protein includes a second label, and wherein the first protecting
group is removed to allow the second biotin molecule to bind with
the second biotin binding protein; binding the third biotin
molecule with a third biotin binding protein, wherein the third
biotin binding protein includes a third label, and wherein the
second protecting group is removed to allow the third biotin
molecule to bind with the third biotin binding protein; and binding
the fourth biotin molecule with a fourth biotin binding protein,
wherein the fourth biotin binding protein includes a fourth label,
and wherein the third protecting group is removed to allow the
fourth biotin molecule to bind with the fourth biotin binding
protein.
41. A method of labeling a plurality of targets with a plurality of
labels, the method comprising: providing a plurality of targets,
wherein the plurality of targets comprises a first target, a second
target, a third target and a fourth target, wherein the first
target includes a first biotin molecule, wherein the first biotin
molecule comprises biotin, wherein the second target includes a
second biotin molecule, wherein the second biotin molecule
comprises desthiobiotin, wherein the third target includes a third
biotin molecule protected by a first protecting group, and wherein
the fourth target includes a fourth biotin molecule protected by a
second protecting group; binding the first biotin molecule with a
first biotin binding protein, wherein the first biotin binding
protein includes a first label, and wherein binding the first
biotin molecule with the first biotin binding protein also binds
the second biotin molecule with the first biotin binding protein;
binding the second biotin molecule with a second biotin binding
protein, wherein the second biotin binding protein includes a
second label, and wherein the first biotin binding protein is
displaced from the second biotin molecule with a biotin solution;
binding the third biotin molecule with a third biotin binding
protein, wherein the third biotin binding protein includes a third
label, and wherein the first protecting group is removed to allow
the third biotin molecule to bind with the third biotin binding
protein; and binding the fourth biotin molecule with a fourth
biotin binding protein, wherein the fourth biotin binding protein
includes a fourth label, and wherein the second protecting group is
removed to allow the fourth biotin molecule to bind with the fourth
biotin binding protein.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 13/329,175,
filed Dec. 16, 2011; which claims the benefit U.S. Provisional
Patent Application No. 61/424,301, filed on Mar. 2, 2011; each of
which are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The presently disclosed compositions and methods provided
herein are for the use of biotin and variants thereof within
labeling and detection schemes employing the associated use of
biotin binding proteins. More specifically, disclosed herein are
compositions and methods with which multi-color labeling and
detection schemes employ one or more variants of biotin and/or one
or more forms of protected biotin.
BACKGROUND OF THE INVENTION
[0003] Biotin has long been employed within the life sciences for a
variety of applications because of several characteristics,
including its small size, relatively easy ability to incorporated
into a variety of materials and substances (i.e., biotinylation),
and its high affinity for certain proteins (e.g., biotin binding
proteins such as avidin, streptavidin and related recombinations,
analogs and derivatives). The small size of biotin enhances its
ability to be incorporated or otherwise label a material or
substance without affecting its biological activity, interaction
with other molecules, etc. Furthermore, through the labeling of
biotin binding proteins with various labels, the high binding
affinity for these proteins with biotin thus facilitates labeling
of targets of interests within assays of many different types. Many
assays in the life sciences utilize two or distinct labels (e.g.,
two types of fluorophores with distinct emission characteristics),
and of these assays, a great number utilize biotin and a biotin
binding protein conjugated with a label as part of the labeling
scheme. However, the use of different binding pairs, often with
inferior binding characteristics, within these labeling schemes to
provide additional label types can lead to undesirable labeling
results and/or increase the difficulty of obtaining accurate
results. Thus, there remains a need for the facilitation of
multi-color labeling approaches where each of the binding pairs for
the labels involved employ a biotin-biotin binding protein
approach.
SUMMARY OF THE INVENTION
[0004] Disclosed herein are compositions and methods for labeling a
plurality of targets with a plurality of labels. Specifically,
compositions and methods are provided for utilizing the high
binding affinity of biotin and its related derivatives and analogs,
such as desthiobiotin, with a wide range of suitable biotin binding
proteins, such as avidin, streptavidin and recombinant versions
thereof. Two or more targets are each biotinylated with a different
biotin molecule, whether the biotin molecule is a different type of
biotin (e.g., desthiobiotin), or whether the biotin molecule is
protected with a suitable protecting group (e.g., photolabile,
acid-labile, and base-labile protecting groups). This
differentiation of the biotins associated with the various targets
at issue allows selective binding with different biotin binding
proteins, where each different biotin binding protein is directly
or indirectly associated with a distinct label. This allows the
various targets, such as nucleic acids, polypeptides, antibodies or
cells, to be differentially labeled even though each of the binding
pairs employs the biotin-biotin binding protein relationship and
its well known characteristics and advantages, such as high binding
affinities and the small size of biotin.
[0005] Furthermore, disclosed herein are compositions and methods
for expanding these labeling schemes from the labeling of two
different targets with two different labels to 4-label assays or
greater. For example, disclosed herein are compositions and methods
for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct labels to be
selected employed within an assay. Labels may be any suitable label
for the assay at issue, including fluorescent, luminescent,
chemiluminescent, light-scattering, and colorimetric labels.
Moreover, the utilization of biotin and biotin derivatives and
analogs can be combined with orthogonal protecting schemes for
biotin to more easily enable 4, 6, 8, etc. label assays.
Particularly suitable protecting groups include DMT and NPOM,
especially NPOM which is linked with the biotin molecule at issue
through an ether linkage, but any suitable photolabile,
acid-labile, base-labile or other protecting group (such as those
removed by hydrogenolysis) which is appropriate for the scheme at
issue may be employed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1(A) illustrates a structural depiction of biotin. FIG.
1(B) illustrates a non-limiting example of biotin with a protecting
group and a cyanoethyl phosphoramidite. FIG. 1(C) and FIG. 1(D)
illustrates two non-limiting examples of potential linkages,
carbamate and ether, between biotin and a protecting group.
[0007] FIG. 2 illustrates non-limiting examples of biotin
protecting groups with alternative linkages.
[0008] FIG. 3 illustrates non-limiting examples of biotin
structural isomers.
[0009] FIG. 4 illustrates a comparison of biotin and
desthiobiotin.
[0010] FIG. 5 illustrates a non-limiting example of a synthesis
scheme to protect biotin and couple the protected biotin with a
cyanoethyl phosphoramidite.
[0011] FIG. 6 illustrates a non-limiting example of a labeling
scheme incorporating the use of biotin with protected biotin.
[0012] FIGS. 7(A)-7(B) illustrate a non-limiting example of a
labeling scheme involving hybridized nucleic acids and ligation
with a set of oligonucleotides which possess biotin or protected
biotin to facilitate multiple label use. FIGS. 7(C)-7(D) illustrate
non-limiting examples of the set of oligonucleotides with either
biotin or protected biotin as used within the depiction of FIGS.
7(A)-7(B).
[0013] FIG. 8 illustrates a non-limiting example of selectively
employing two labels within the example depicted within FIGS.
7(A)-7(D).
[0014] FIG. 9 illustrates non-limiting examples of alternative
protecting groups.
[0015] FIG. 10 illustrates a non-limiting example of a labeling
scheme incorporating the use of biotin and desthiobiotin.
[0016] FIGS. 11(A)-11(B) illustrate a non-limiting example of a
labeling scheme involving hybridized nucleic acids and ligation
with a set of oligonucleotides which possess biotin or
desthiobiotin to facilitate multiple label use. FIGS. 11(C)-11(D)
illustrate non-limiting examples of the set of oligonucleotides
with either biotin or desthiobiotin as used within the depiction of
FIGS. 11(A)-11(B).
[0017] FIG. 12 illustrates a non-limiting example of selectively
employing two labels within the example depicted within FIGS.
11(A)-11(D).
[0018] FIGS. 13(A)-13(B) contain images of arrays utilized to
measure the ability of a DMT protecting group to prevent a
streptavidin fluorophore conjugate from binding with the protected
biotin.
[0019] FIGS. 14(A)-14(C) contain images of arrays utilized to
measure the ability of selected acids at different pH values to
remove DMT protecting groups from biotin and allow desired binding
with a streptavidin fluorophore conjugate.
[0020] FIGS. 15(A)-15(C) contain images of arrays utilized to
measure the ability of desthiobiotin to bind with a streptavidin
fluorophore conjugate, have the streptavidin fluorophore conjugate
be displaced by a biotin solution, and rebind with a streptavidin
fluorophore conjugate.
DETAILED DESCRIPTION
[0021] The present invention has many preferred embodiments and
relies on many patents, patent applications and other references
for details known to those of ordinary skill in the art to which
the invention pertains. Therefore, when a reference, such as a
patent, patent application, and other publication is cited or
otherwise mentioned within any section of this specification, it
should be understood that the reference is incorporated by
reference in its entirety for all purposes as well as for the
proposition that is recited and/or the precise context in which the
reference is cited.
[0022] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
organic chemistry, polymer technology, molecular biology (including
recombinant techniques), cell biology, biochemistry, and
immunology, which are within the skill of the art. Such
conventional techniques may include polymer array synthesis,
hybridization, ligation, and detection of hybridization using a
label. Specific illustrations of suitable techniques can be had by
reference to the embodiments described herein. However, other
equivalent conventional procedures can, of course, also be used.
Such conventional techniques and descriptions can be found in
standard laboratory manuals such as Genome Analysis: A Laboratory
Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual,
Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and
Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor
Laboratory Press), Stryer, Biochemistry, 4.sup.th Ed., W.H. Freeman
& Company (1995), Gait, "Oligonucleotide Synthesis: A Practical
Approach," IRL Press, London (1984), Nelson and Cox, Lehninger,
Principles of Biochemistry 3.sup.rd Ed., W.H. Freeman Pub., New
York, N.Y. (2000), and Berg et al., Biochemistry, 5.sup.th Ed.,
W.H. Freeman Pub., New York, N.Y. (2002), all of which are herein
expressly incorporated by reference in their entirety for all
purposes.
[0023] Embodiments of the present invention may utilize enzymatic
activities. A variety of enzymes are well known, have been
characterized and many are commercially available from one or more
supplier. For a review of enzyme activities commonly used in
molecular biology see, for example, Rittie and Perbal, J. Cell
Commun. Signal., 2: 25-45 (2008). Exemplary enzymes include DNA
dependent DNA polymerases (such as those shown in Table 1 of Rittie
and Perbal), RNA dependent DNA polymerase (see Table 2 of Rittie
and Perbal), RNA polymerases, ligases (see Table 3 of Rittie and
Perbal), enzymes for phosphate transfer and removal (see Table 4 of
Rittie and Perbal), nucleases (see Table 5 of Rittie and Perbal),
and methylases.
[0024] Nucleic acid arrays may be employed in various embodiments,
with commercially available arrays including GeneChip.RTM. and
Axiom.RTM. arrays from Affymetrix, Inc. (Santa Clara, Calif.),
Infinium.RTM. and GoldenGate.RTM. arrays from Illumina, Inc. (San
Diego, Calif.), NimbleGen.RTM. arrays from Roche NimbleGen, Inc.
(Madison, Wis.), Agilent.RTM. arrays from Agilent Technologies,
Inc. (Santa Clara, Calif.). Such arrays may be employed within, for
example, molecular diagnostics, copy number analysis, genome-wide
genotyping, drug metabolism analysis, molecular cytogenetics,
resequencing analysis, targeted genotyping analysis, expression
analysis, gene regulation analysis, miRNA analysis,
whole-transcript expression analysis and profiling, and other uses
known in the art. Various methods of gene expression monitoring and
profiling are described in, for example, U.S. Pat. Nos. 5,800,992;
6,013,449; 6,020,135; 6,033,860; 6,040,138; 6,177,248 and
6,309,822. Genotyping methods are disclosed in, for instance, U.S.
Pat. Nos. 5,856,092; 6,300,063; 5,858,659; 6,284,460; 6,361,947;
6,368,799; 6,333,179 and 6,872,529. Other uses of arrays are
described in, for example, U.S. Pat. Nos. 5,871,928; 5,902,723;
6,045,996; 5,541,061 and 6,197,506. Methods and techniques
applicable to polymer array synthesis have been described in the
art, for example, in International Patent Publication Nos. WO
99/36760; WO 00/58516 and WO 01/58593; and U.S. Pat. Nos.
5,143,854; 5,242,974; 5,252,743; 5,324,633; 5,384,261; 5,405,783;
5,424,186; 5,451,683; 5,482,867; 5,491,074; 5,527,681; 5,550,215;
5,571,639; 5,578,832; 5,593,839; 5,599,695; 5,624,711; 5,631,734;
5,795,716; 5,831,070; 5,837,832; 5,856,101; 5,858,659; 5,936,324;
5,968,740; 5,974,164; 5,981,185; 5,981,956; 6,025,601; 6,033,860;
6,040,193; 6,090,555; 6,136,269; 6,269,846; and 6,428,752, all of
which are incorporated herein by reference in their entireties for
all purposes.
[0025] The synthesis of oligonucleotides on the surface of a
substrate may be carried out using light directed methods as
described in., e.g., U.S. Pat. Nos. 5,143,854 and 5,384,261 and PCT
Publication No. WO 92/10092, or mechanical synthesis methods as
described in U.S. Pat. Nos. 5,384,261, 6,040,193 and PCT
Publication No. 93/09668, each of which is incorporated herein by
reference. In particular, these light-directed or photolithographic
synthesis methods involve a photolysis step and a chemistry step.
The substrate surface, prepared as described herein, comprises
functional groups on its surface. These functional groups are
protected by photolabile protecting groups. During the photolysis
step, portions of the surface of the substrate are exposed to light
or other activators to activate the functional groups within those
portions, e.g., to remove photolabile groups. The substrate is then
subjected to a chemistry step in which chemical monomers that are
photoprotected at least one functional group are then contacted
with the surface of the substrate. These monomers bind to the
activated portion of the substrate through an unprotected
functional group. Repetitions of the activation and coupling steps
may be employed in sets of preselected regions, which may overlap,
at least in part, with the first set of preselected regions, to
create an array of polymers with different sequences within the
regions of the substrate.
[0026] Preparation methods for samples of nucleic acids are well
known in the art. Certain methods may implement amplification by a
variety of mechanisms, some of which may employ PCR. (See, e.g.,
PCR Technology: Principles and Applications for DNA Amplification,
Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992; PCR Protocols: A
Guide to Methods and Applications, Eds. Innis, et al., Academic
Press, San Diego, Calif., 1990; Mattila et al., Nucleic Acids Res.,
19: 4967 (1991); Eckert et al., PCR Methods and Applications, 1:17
(1991); PCR, Eds. McPherson et al., IRL Press, Oxford (1991); and
U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; 4,965,188 and
5,333,675, each of which is incorporated herein by reference in
their entireties for all purposes. Isothermal amplification methods
have also been developed. One of them is known as Strand
Displacement Amplification (SDA). SDA combines the ability of a
restriction endonuclease to nick the unmodified strand of its
target DNA and the action of an exonuclease-deficient DNA
polymerase to extend the 3' end at the nick and displace the
downstream DNA strand. The displaced strand serves as a template
for an antisense reaction and vice versa, resulting in exponential
amplification of the target DNA (See, e.g., U.S. Pat. Nos.
5,455,166 and 5,470,723). Another isothermal amplification system,
Transcription-Mediated Amplification (TMA), utilizes the function
of an RNA polymerase to make RNA from a promoter engineered in the
primer region, and a reverse transcriptase, to produce DNA from the
RNA templates. This RNA amplification technology has been further
improved by introducing a third enzymatic activity, RNase H, to
remove the RNA from cDNA without the heat-denaturing step. Thus the
thermo-cycling step has been eliminated, generating an isothermal
amplification method named Self-Sustained Sequence Replication
(3SR) (See, e.g., Guatelli et al., Proc. Natl. Acad. Sci. USA
87:1874-1878 (1990)). The starting material for TMA and 3SR is RNA
molecules. Many other amplification techniques, and sample
preparation methods in general, are known to those of skill in the
art.
[0027] Other suitable sample amplification methods include the
ligase chain reaction (LCR) (see, e.g., Wu and Wallace, Genomics,
4: 560 (1989); Landegren et al., Science, 241: 1077 (1988); and
Barringer et al., Gene, 89: 117 (1990)), transcription
amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86: 1173
(1989) and WO 88/10315), self-sustained sequence replication
(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87: 1874 (1990) and WO
90/06995), selective amplification of target polynucleotide
sequences (e.g., U.S. Pat. No. 6,410,276), consensus sequence
primed polymerase chain reaction (CP-PCR) (U.S. Pat. No.
4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR)
(U.S. Pat. Nos. 5,413,909 and 5,861,245) and nucleic acid based
sequence amplification (NABSA). (See also, U.S. Pat. Nos.
5,409,818; 5,554,517; and 6,063,603, each of which is incorporated
herein by reference). Other amplification methods that may be used
are described in, for instance, U.S. Pat. Nos. 6,582,938;
5,242,794; 5,494,810 and 4,988,617, each of which is incorporated
herein by reference. Additional methods of sample preparation and
techniques for reducing the complexity of a nucleic sample are
described in Dong et al., Genome Research, 11:1418 (2001), U.S.
Pat. Nos. 6,361,947; 6,391,592; 6,632,611; 6,872,529 and
6,958,225.
[0028] Another amplification method, Rolling Circle Amplification
(RCA), generates multiple copies of a sequence for the use in in
vitro DNA amplification adapted from in vivo rolling circle DNA
replication (See, e.g., Fire and Xu, Proc. Natl. Acad. Sci. USA
92:4641-4645 (1995); Lui, et al., J. Am. Chem. Soc. 118:1587-1594
(1996); Lizardi, et al., Nature Genetics 19: 225-232 (1998), U.S.
Pat. Nos. 5,714,320 and 6,235,502). In this reaction, a DNA
polymerase extends a primer on a circular template generating
tandemly linked copies of the complementary sequence of the
template. RCA has been further developed in a technique, named
Multiple Displacement Amplification (MDA), which generates a highly
uniform representation in whole genome amplification (See, e.g.,
Dean et. al., Proc. Natl. Acad. Sci. USA 99:5261-5266 (2002)).
[0029] Methods for conducting polynucleotide hybridization assays
have been well developed in the art. Hybridization assay procedures
and conditions will vary depending on the application and are
selected in accordance with known general binding methods,
including those referred to in Maniatis et al., Molecular Cloning:
A Laboratory Manual, 2d Ed., Cold Spring Harbor, N.Y, (1989);
Berger and Kimmel, Methods in Enzymology, Guide to Molecular
Cloning Techniques, Vol. 152, Academic Press, Inc., San Diego,
Calif. (1987); Young and Davism, Proc. Nat'l. Acad. Sci., 80: 1194
(1983). Methods and apparatus for performing repeated and
controlled hybridization reactions have been described in, for
example, U.S. Pat. Nos. 5,871,928; 5,874,219; 6,045,996; 6,386,749
and 6,391,623, each of which are incorporated herein by reference.
The invention also contemplates signal detection of hybridization
between ligands in certain embodiments. (See, e.g., U.S. Pat. Nos.
5,143,854; 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956;
6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803 and
6,225,625, each of which is hereby incorporated by reference in its
entirety for all purposes). Many other related techniques and
developments thereof are also known in the art.
[0030] Described herein are various chemical structures for use
within various embodiments. As described or depicted herein, an
alkyl group refers to a straight or branched hydrocarbon chain
containing the specified number of carbon atoms. If alkyl is used
without reference to a number of carbon atoms, it is to be
understood to refer to a C.sub.1-C.sub.10 alkyl. For example,
C.sub.1-10 alkyl refers to a straight or branched alkyl containing
at least 1, and at most 10, carbon atoms. Examples of alkyls
include, but are not limited to, methyl, ethyl, n-propyl, n-butyl,
n-pentyl, isobutyl, isopropyl, t-butyl, hexyl, heptyl, octyl, nonyl
and decyl.
[0031] Aryl refers to an aromatic monovalent carboxylic radical
having a single ring (e.g., phenyl) or two condensed rings (e.g.,
naphthyl), which can optionally be mono-, di-, or tri-substituted,
independently, with alkyl, lower-alkyl, cycloalkyl,
ydroxylower-alkyl, aminoloweralkyl, hydroxyl, thiol, amino, halo,
nitro, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino,
di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl,
hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl,
lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl,
lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl. Cycloalkyl
groups generally refer to a non-aromatic monocyclic hydrocarbon
ring of 3 to 8 carbon atoms such as, for example, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.
[0032] Alternatively, two adjacent positions of the aromatic ring
may be substituted with a methylenedioxy or ethylenedioxy group.
Heteroaromatic refers to an aromatic monovalent mono- or
poly-cyclic radical having at least one heteroatom within the ring,
e.g., nitrogen, oxygen or sulfur, wherein the aromatic ring can
optionally be mono-, di- or tri-substituted, independently, with
alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl,
aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro,
lower-alkylthio, loweralkoxy, mono-lower-alkylamino,
di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl,
hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl,
lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl,
loweralkylcarbamoyl, and di-lower-alkylcarbamoyl. For example,
typical heteroaryl groups with one or more nitrogen atoms are
tetrazoyl, pyridyl (e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl),
pyrrolyl (e.g., 2-pyrrolyl, 2-(N-alkyl)pyrrolyl), pyridazinyl,
quinolyl (e.g. 2-quinolyl, 3-quinolyl etc.), imidazolyl,
isoquinolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridonyl or
pyridazinonyl; typical oxygen heteroaryl radicals with an oxygen
atom are 2-furyl, 3-furyl or benzofuranyl; typical sulfur
heteroaryl radicals are thienyl, and benzothienyl; typical mixed
heteroatom heteroaryl radicals are furazanyl and
phenothiazinyl.
[0033] Substitution of chemical groups may, in certain
circumstances, be optional, and thus optionally substituted refers
to the presence or lack thereof of a substituent on the group being
defined. When substitution is present the group may be mono-, di-
or tri-substituted, independently, with alkyl, lower-alkyl,
cycloalkyl, hydroxylower-alkyl, aminoloweralkyl, hydroxyl, thiol,
amino, halo, nitro, lower-alkylthio, lower-alkoxy,
mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl,
lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl,
lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano,
tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and
di-lower-alkylcarbamoyl. Typically, electron-donating substituents
such as alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl,
aminoloweralkyl, hydroxyl, thiol, amino, halo, lower-alkylthio,
lower-alkoxy, mono-lower-alkylamino and di-lower-alkylamino are
often preferable.
[0034] Substituted alkyls generally refer to alkyl radicals wherein
at least one hydrogen is replaced by one more substituents such as,
but not limited to, hydroxy, alkoxy, aryl (for example, phenyl),
heterocycle, halogen, trifluoromethyl, pentafluoroethyl, cyano,
cyanomethyl, nitro, amino, amide (e.g., --C(O)NH--R where R is an
alkyl such as methyl), amidine, amido (e.g., --NHC(O)--R where R is
an alkyl such as methyl), carboxamide, carbamate, carbonate, ester,
alkoxyester (e.g., --C(O)O--R where R is an alkyl such as methyl)
and acyloxyester (e.g., --OC(O)--R where R is an alkyl such as
methyl), or two hydrogens on a single carbon is replaced with
oxygen to provide a carbonyl group. T
[0035] Substituted cycloalkyls generally refer to a cycloalkyl
group which further bears one or more substituents as set forth
herein, such as, but not limited to, hydroxy, alkoxy, aryl (for
example, phenyl), heterocycle, halogen, trifluoromethyl,
pentafluoroethyl, cyano, cyanomethyl, nitro, amino, amide (e.g.,
--C(O)NH--R where R is an alkyl such as methyl), amidine, amido
(e.g., --NHC(O)--R where R is an alkyl such as methyl),
carboxamide, carbamate, carbonate, ester, alkoxyester (e.g.,
--C(O)O--R where R is an alkyl such as methyl) and acyloxyester
(e.g., --OC(O)--R where R is an alkyl such as methyl), or two
hydrogen atoms on a single carbon is replaced with oxygen to
provide a carbonyl group.
[0036] The practice of embodiments of the present invention may
also employ conventional software methods and systems. Computer
software products utilized with embodiments of the present
invention generally include computer readable medium having
computer-executable instructions for performing various steps
directly or indirectly associated with aspects of the present
invention. Suitable computer readable medium include floppy disk,
CD-ROM/DVD/DVD-ROM, hard-disk drive (e.g., utilized locally and/or
over a network), flash memory, ROM/RAM, magnetic tapes and etc. The
computer executable instructions may be written in a suitable
computer language or combination of several languages. Basic
computational biology methods are described in, e.g. Setubal and
Meidanis et al., Introduction to Computational Biology Methods, PWS
Publishing Company, Boston (1997), Salzberg, Searles, Kasif, (Ed.),
Computational Methods in Molecular Biology, Elsevier, Amsterdam
(1998), Rashidi and Buehler, Bioinformatics Basics: Application in
Biological Science and Medicine, CRC Press, London (2000) and
Ouelette and Bzevanis Bioinformatics: A Practical Guide for
Analysis of Gene and Proteins, Wiley & Sons, Inc., 2.sup.nd ed.
(2001). Embodiments may also make use of various computer program
products and software for a variety of purposes, such as probe
design, management of data, analysis, and instrument operation.
(See, e.g., U.S. Pat. Nos. 5,593,839; 5,795,716; 5,733,729;
5,974,164; 6,066,454; 6,090,555; 6,185,561; 6,188,783; 6,223,127;
6,229,911 and 6,308,170). Additionally, embodiments may include
methods for providing biological information over networks such as
the internet, as disclosed in, for instance, U.S. Patent
Application Publication Nos. 2003/0097222; 2002/0183936;
2003/0100995; 2003/0120432; 2004/0002818; 2004/0126840 and
2004/0049354.
I. DEFINITIONS
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. The
following definitions supplement those in the art, are directed to
the current application, and are not to be imputed to any related
or unrelated case, e.g., to any commonly owned patent or patent
application. Accordingly, the terminology used herein is for the
purpose of describing particular embodiments only, and is not
intended to be limiting.
[0038] Throughout this disclosure, various aspects of the invention
may be presented in a range format. It should be understood that
when a description is provided in range format, this is merely for
convenience and brevity, and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible sub-ranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed sub-ranges such as from 1 to 2, from 1
to 2.5, from 1 to 3, from 1 to 3.5, from 1 to 4, from 1 to 4.5,
from 1 to 5, from 1 to 5.5, from 2 to 4, from 2 to 6, and from 3 to
6 for example, as well as individual numbers within that range, for
example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6. This
applies regardless of the breadth of the range.
[0039] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a molecule" includes a plurality of such molecules,
and the like.
[0040] The term "about" as used herein indicates the value of a
given quantity varies by +/-10% of the value, or optionally +/-5%
of the value, or in some embodiments, by +/-1% of the value so
described.
[0041] The term "biotin" as used herein refers to the naturally
occurring vitamin and also analog, derivative, and other modified
forms which are suitable for the application in which the term is
employed, whether or not one or more modified forms are expressly
mentioned.
[0042] The term "biotin binding protein" as used herein refers to
any suitable protein capable of binding with biotin with a binding
affinity and specificity sufficiently high for the application in
which the term is employed, and which thus may vary depending on
the particular application. This reference applies whether or not
one or more modified forms are expressly mentioned. Non-limiting
examples include avidin, streptavidin, analogs, derivatives and
other modified forms thereof.
[0043] The term "label" as used herein refers to a molecule or
combination of molecules which facilitate detection of another
molecule or combination of molecules. The label may be a detectable
chemical or biochemical moiety or a signal obtained from an
enzyme-linked assay. The label molecule(s) can be applied directly
to the label target or indirectly through the use of two or more
sets of molecules.
[0044] The terms "polynucleotide" and "nucleic acid" as used herein
are used interchangeably and encompass any physical string of
monomer units that can correspond to a string of nucleotides,
including a polymer of nucleotides (e.g., a typical DNA or RNA
polymer), peptide nucleic acids (PNAs), modified oligonucleotides
(e.g., oligonucleotides comprising nucleotides that are not typical
to biological RNA or DNA, such as 2'-O-methylated oligonucleotides,
LNA, etc.), and the like. The nucleotides of the polynucleotide can
be deoxyribonucleotides, ribonucleotides or nucleotide analogs, can
be natural or non-natural, and can be unsubstituted, unmodified,
substituted or modified. The nucleotides can be linked by
phosphodiester bonds, or by phosphorothioate linkages,
methylphosphonate linkages, boranophosphate linkages, or the like.
The polynucleotide can optionally further comprise non-nucleotide
elements such as labels, quenchers, blocking probes, or the like.
The polynucleotide can be, e.g., single-stranded or
double-stranded.
[0045] A "polynucleotide sequence" or "nucleotide sequence" or
"nucleic acid sequence" as used herein are used interchangeably and
encompass polymers of nucleotides (e.g., an oligonucleotide, a DNA,
a nucleic acid, etc.) or a character string representing a
nucleotide polymer, depending on context. From any specified
polynucleotide sequence, either the given nucleic acid or the
complementary polynucleotide sequence (e.g., the complementary
nucleic acid) can be determined.
[0046] A "protecting group" or "protective group" as used
interchangeably herein refers to any moiety or molecule designed to
block a reactive site in a molecule. A protecting group may be a
material which is chemically bound to a reactive functional group
on a monomer unit or polymer and which may be removed upon
selective exposure to an activator such as a chemical activator, or
another activator, such as electromagnetic radiation or light
(e.g., ultraviolet or visible light). Protecting groups that are
removable upon exposure to electromagnetic radiation (e.g.,
ultraviolet or visible light) are referred to herein as protecting
groups that are "photolabile" or "photolyzable." Other protecting
groups, however, employ a different mechanism for deprotection,
such as the use of acid or base to deprotect the relevant molecule.
Still other protecting groups employ hydrogenolysis for
deprotection. Examples of suitable protecting groups for certain
embodiments include those described in "Protecting Groups," Phillip
J. Kocie ski, Thieme (3rd Ed. 2005); "Greene's Protecting Groups in
Organic Synthesis," Peter G. M. Wuts and Theodora W. Greene,
Wiley-Interscience (4th Ed. 2006); "Handbook of Synthetic
Photochemistry," Angelo Albini and Maurizio Fagnoni (Eds.),
Wiley-VCH (2010); U.S. Pat. No. 6,147,205 to McGall et al.; U.S.
Pat. No. 7,547,775 to Kuimelis et al.; U.S. Patent Application
Publication No. 2011/0028350; U.S. Patent Application Publication
No. 2003/0040618 to McGall et al.; and U.S. Patent Application
Publication No. 2006/0147969 to Kuimelis et al., all of which are
incorporated herein by reference in their entireties for all
purposes, and particularly for their disclosed protecting groups
and related methods of manufacture and use. The proper selection of
protecting groups for a particular synthesis is governed by the
overall methods employed in the synthesis.
[0047] The term "support" or "substrate" as used interchangeably
herein refers to a material or group of materials, comprising one
or more components, with which one or more molecules are directly
or indirectly bound, attached, synthesized upon, linked, or
otherwise associated. A support may be constructed from materials
that are biological, non-biological, organic, inorganic or a
combination of these. A support may be in any suitable size or
configuration based upon its use within a particular
embodiment.
[0048] The term "target" as used herein refers to a molecule of
interest within an assay. Targets may be naturally occurring or
synthetic, or a combination. Targets may be unaltered (e.g.,
utilized directly within the organism or a sample thereof), or
alternated in a manner appropriate for the assay (e.g., purified,
amplified, filtered). Targets may be bound through a suitable means
to a binding member within certain assays. Non-limiting examples of
targets include, but are not restricted to, antibodies or fragments
thereof, cell membrane receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, oligonucleotides,
nucleic acids, peptides, cofactors, lectins, sugars,
polysaccharides, cells, cellular membranes, and organelles. Target
may be any suitable size depending on the assay, as nucleic acid
targets may merely be a single nucleotide (or a component thereof,
such as the base, sugar or phosphate group) and polypeptide targets
may merely be a single amino acid (or a component thereof, such as
the amine, carboxylic acid or side-chain group).
II. SPECIFIC EMBODIMENTS
Creation of Functionalized Substrates
[0049] Various embodiments disclosed herein may be utilized in
association with substrates which have one or more surfaces
functionalized with an appropriate functional group. Non-limiting
examples of functional groups include aldehyde, alkene, alkyne,
amine, aminooxy, azide, carbonyl, carboxyl, carboxylate, disulfide,
halogen, hydrazine, hydroxyl, isocyanate, isothiocyanate, sulfate,
sulfonate, thiol and thiocarboxyl functional groups. Other suitable
functional groups may also be utilized within certain embodiments,
such as modified forms of the preceding examples (e.g., activated
or protected forms). Various applications may, for example,
covalently attach these functionalized silicon compounds to a
surface of the substrate, thus forming a functionalized surfaced
for subsequent use. For example, a silicon compound with hydroxyl
functional groups may be attached to a silicon dioxide substrate,
such as a wafer, slide, bead, or microparticle, to provide a
functionalized surface. This functionalized surface may
subsequently be utilized within the attachment of other molecules,
such as nucleic acid monomers, peptide monomers, polynucleotides,
and polypeptides, to the surface. For instance, such a surface may
be utilized within the creation of a nucleic acid array, such as a
high density array, through the covalent attachment and
immobilization of nucleic acids or nucleic acid monomers to the
functionalized surface.
[0050] As used herein, a support or a substrate refers to any
suitable material or combination of materials onto which molecules,
such as the non-limiting examples recited above of polynucleotides
or polypeptides, are synthesized, attached, or otherwise bound. In
addition, the substrate may comprise a single component of one or
more materials, or multiple components which are utilized together
(e.g., assembled into a single substrate component). In many
embodiments, the material is rigid or at least semi-rigid, and
often has at least one surface that is substantially flat or
planar. However, other surfaces on the substrate may not be flat or
planar, as may be desirable for manufacturing or subsequent use of
the substrate. Furthermore, other embodiments do not utilize flat
or planar surfaces. For example, certain embodiments utilize
substrates with a surface that possesses physical features such as
wells, raised regions, etched trenches, or other features that may
be inherent to the substrate material(s). Other embodiments may
employ substrate conformations such as particles, microparticles,
strands, precipitates, gels, sheets, tubes, spheres, containers,
capillaries, pads, slices, films, plates, slides or other
conformations known in the art. In other embodiments, substrate
conformations of tubes, capillaries or microcapillaries are
employed. These embodiments can offer higher surface area to volume
ratios compared to certain other embodiments while providing
benefits such as reagent reduction and improving thermal transfer
efficiency. In addition, some embodiments will utilize physical
features in association with the substrate that are only temporary,
such as beads that are attached or otherwise associated with a
substrate surface and which are released during the overall
manufacturing process which utilizes the substrate.
[0051] The support or substrate may comprise one or more components
of any suitable material(s), including the non-limiting examples of
fused silica, fused quartz, glass, Si, SiO.sub.2, SiN.sub.4, other
silicon based materials, Ge, GeAs, GaP, polyvinylidene fluoride,
polycarbonate, other polymers, and combinations of these and other
suitable materials known in the art. The suitability of substrate
material(s) will depend on various factors, such the manufacturing
approach to be utilized, assay conditions to which the substrate
will be exposed, and other factors. For example, if
photolithography is employed for in situ synthesis of
oligonucleotides on a functionalized surface of a substrate, then
silicon based materials, such as those utilized in the
semiconductor and microprocessor industries, may be appropriate for
certain embodiments. The appropriateness of material(s) will depend
on multiple factors. For example, within the previous example of
silicon based substrate materials, such as silicon dioxide, this is
utilized with photolithography, certain embodiments may favor a
substrate that is transparent or substantially transparent to
enable efficient and effective illumination from either side of the
substrate (e.g., the side of the substrate with the functionalized
surface, or the opposite side of the substrate). A non-limiting
example of such a substrate is a wafer of fused silica that is
utilized as the substrate. In one particular non-limiting
embodiment, the wafer ranges in size from about 1'' by about 1'' to
about 12'' by about 12'', and a thickness from about 0.5 mm to
about 5.0 mm. Such a wafer may be subdivided during the
manufacturing process to create multiple subunits, such as the
creation of multiple arrays by dicing the wafer into subdivisions.
For instance, the diced subunits may measure from about 0.2 cm by
about 0.2 cm to about 5.0 cm by about 5 cm. A non-limiting example
is a wafer that is 5'' by 5'' that is diced into 49 subunits of
1.28 cm by 1.28 cm. The size of the starting wafer utilized as the
substrate and the size of the subunits will depend on various
factors, such as the manufacturing approach employed, the desired
characteristics of the final substrate subunits, and assay
requirements. See, e.g., U.S. Pat. Nos. 5,143,854; 5,959,098; and
7,332,273, all of which are expressly incorporated by reference
herein for all purposes.
[0052] Other suitable substrate formats may be utilized, as are
appropriate for the manufacturing approach and assay at issue. For
example, microparticles may be utilized in certain embodiments.
Non-limiting examples of microparticles, methods of manufacturing
them, and methods and systems for detecting them and employing them
in various assays can be found in, for example, U.S. Pat. Nos.
7,745,091 and 7,745,092 to True; U.S. Patent Application
Publication No. 2010/0297448 to True et al.; and U.S. Patent
Application Publication Nos. 2010/0227279, 2010/0227770, and
2009/0149340 to True, all of which are expressly incorporated by
reference herein for all purposes.
[0053] Another substrate format which some embodiments employ is an
aerogel, such as a silica or carbon based aerogel which is utilized
as the substrate, a portion of a substrate, as a component of a
substrate, etc. Aerogel substrates may be used as free standing
substrates or as a surface coating for another rigid substrate.
Aerogel substrates provide the advantage of large surface area for
polymer synthesis. For example, a 1 cm.sup.2 portion of an aerogel
substrate can possess a total useful surface area of 100 to 1000
cm.sup.2 (e.g., 400 to 1000 m.sup.2/g). Such aerogel substrates may
be prepared by any suitable method known in the art. For example,
in one embodiment, a silica aerogel substrate is prepared by the
base catalyzed polymerization of (MeO).sub.4Si or (EtO).sub.4Si in
an ethanol/water solution at room temperature. Porosity and other
applicable properties may be adjusted by any suitable change to
manufacturing known in the art, such as appropriate alterations to
reaction conditions.
[0054] The derivatization of substrates with functional groups may
occur through any suitable means known in the art. For example,
silanation reagents for the silanation of substrates are well
known. Such silanation can, for example, prepare substrates with
functional groups that can be further derivatized or otherwise
utilized for various purposes, such as providing functional groups
with which to prepare polynucleotide or polypeptide arrays on the
substrate. For example,
N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (PCR Inc.,
Gainesville, Fla. and Gelest Inc., Tullytown, Pa.) has been used to
silylate a glass substrate prior to photochemical synthesis of
arrays of oligonucleotides on the substrate, as described in McGall
et al., J. Am. Chem. Soc., 119: 5081-5090 (1997), which is
incorporate herein by reference in its entirety. Hydroxyalkylsilyl
compounds have been used to prepare hydroxyalkylated substrates,
such as fused silica wafers. N,N-Bis-(hydroxyethyl)
aminopropyl-triethoxysilane (BHAPTES) has been used to treat glass
substrates to permit the synthesis of high-density oligonucleotide
arrays. McGall et al., Proc. Natl. Acad. Sci., 93: 13555-13560
(1996); and Pease et al., Proc. Natl. Acad. Sci., 91: 5022-5026
(1994), which are both incorporated herein by reference in their
entireties. Acetoxypropyl-triethoxysilane has been used to treat
glass substrates to prepare them for oligonucleotide array
synthesis, as described in International Patent Publication No. WO
97/39151, which is incorporated herein by reference in its
entirety. 3-glycidoxy propyltrimethoxysilane has been used to treat
a glass substrate to provide a linker for the synthesis of
oligonucleotides, see, e.g., EP Patent No. 0 368 279, which is
incorporated herein by reference in its entirety. Many other
silanation reagents and approaches for employing them are known in
the art, and are appropriate for use with various embodiments
disclosed herein. Certain silanation reagents are purposefully
sterically hindered for the creation of more stable surfaced bonded
silicon compounds. See, e.g., Kirkland et al., Anal. Chem. 61: 2-11
(1989); and Schneider et al., Synthesis, 1027-1031 (1990). However,
such sterically hindered reagents are often more difficult to bond
to the substrate as their hindered nature reduces their
reactiveness with the substrate.
[0055] Additionally, silanes can be prepared which have protected
or masked functional groups. Such silanes can be readily purified
by, e.g., distillation, and employed in suitable silanation
methods, such as gas-phase deposition of a surface of a substrate.
After appropriate silanation of a substrate with these silanes, the
functional groups can be deprotected through an appropriate manner
based upon the type and design of the protection. For example, a
silane such as acetoxyalkylsilane, acetoxyethyltrichlorosilane, or
acetoxypropyltrimethoxysilane can be deprotected after application
using an appropriate method, such as vapor phase ammonia and
methylamine or liquid phase aqueous or ethanolic ammonia and
alkylamines. Many other silanes may be employed and appropriately
deprotected, such as epoxyalkylsilanes (e.g.,
glycidoxypropyltrimethoxysilane) deprotected using vapor phase HCl
or trifluoroacetic acid, or alternatively with liquid phase dilute
HCl.
[0056] Following application of the silane reagents to form a
silane layer, the silanated substrate may be baked to polymerize
the silanes on the surface of the substrate and improve the
reaction between the silane reagent and the substrate surface. The
characteristics of the baking will vary depending on the particular
embodiment, the silanes at issue, the substrate and the desired
characteristics of the resulting substrate. A non-limiting example
of baking conditions suitable for certain embodiments include a
temperature in the range of from 90.degree. C. to 120.degree. C.,
for example 110.degree. C., and a time period of from about 1
minute to about 120 minutes, for example for 60 minutes. This
non-limiting example should not be construed to be limiting, as
other embodiments may employ conditions within these ranges (e.g.,
temperatures of 95.degree. C., 110.degree. C.; time periods of 5
minutes, 30 minutes, or 90 minutes) as well as conditions outside
of these ranges (e.g., temperatures of 80.degree. C., 125.degree.
C.; time periods of 30 seconds, 145 minutes, 160 minutes).
[0057] The silanation reagents may be brought into contact with a
surface of a substrate through any suitable method. Non-limiting
examples of suitable methods include vapor deposition or spray
methods. These methods may involve, for example, the volatilization
or atomization of a silane solution into a gas phase or spray,
followed by deposition of the gas phase or spray upon the surface
of the substrate, often by ambient exposure of the surface of the
substrate to the gas phase or spray. Vapor deposition typically
results in a more even application of the derivatization solution
than simply immersing the substrate into the solution. The efficacy
of the derivatization process, e.g., the density and uniformity of
functional groups on the substrate surface, may generally be
assessed by adding a fluorophore which binds to the functional
groups. For example, a phosphoramidite with a suitable fluorescent
label can be reacted with the functional groups, and the resulting
fluorescent across the surface of the substrate analyzed to assess
efficacy.
Creation of Polymer Arrays
[0058] Creation of polymer arrays of different biological polymer
sequences, such as nucleic acid and polypeptide arrays, through a
variety of techniques is well known. See, e.g., U.S. Pat. No.
5,143,854 to Pirrung et al.; U.S. Pat. No. 5,744,305 to Fodor et
al.; U.S. Pat. No. 7,332,273 to Trulson et al.; U.S. Pat. No.
6,242,266 to Schleifer et al.; U.S. Pat. No. 6,375,903 to Cerrina
et al.; U.S. Pat. No. 5,436,327 to Southern et al.; U.S. Pat. No.
5,474,796 to Brennan; U.S. Pat. No. 5,658,802 to Hayes et al.; U.S.
Pat. No. 5,770,151 to Roach et al.; U.S. Pat. No. 5,807,522 to
Brown et al.; U.S. Pat. No. 5,981,733 to Gamble et al.; and U.S.
Pat. No. 6,101,946 to Martinsky, all of which are expressly
incorporated herein by reference for all purposes. Such arrays may
contain hundreds, thousands, or millions of different
polynucleotide or polypeptide sequences, depending upon, for
example, the abilities of the particular manufacturing technique at
issue with respect to feature density, the size of the relevant
solid support of silicon, glass, or other material, the desired
characteristics of the relevant assay, and other factors. Within
the non-limiting example of nucleic acid arrays, probes of
polynucleotides may range from, for example, 10-200 nucleotides in
length., such as 10, 15, 25, 30, 35, 40, 45, 50, 65, 70, 75, 100,
125, 150, or 200 nucleotides. This range should not be construed to
be limiting, as depending on the manufacturing approach, assay
characteristics, and many other factors, nucleic acid arrays may
include probes within this range not explicitly mentioned above,
such as 28, 49, or 79 nucleotides in length, or probes outside of
this range, such as 9 nucleotides or 250 nucleotides.
[0059] Certain embodiments may utilize arrays created through Very
Large Scale Immobilized Polymer Synthesis (VLSIPS) technology for
synthesizing oligonucleotides and oligonucleotide analogues on
substrates. The oligonucleotide is typically linked to the
substrate via the 3'-hydroxyl group of the oligonucleotide and a
functional group on the substrate which results in the formation of
an ether, ester, carbamate or phosphate ester linkage. Nucleotide
or oligonucleotide analogues are attached to the solid support via
carbon-carbon bonds using, for example, supports having
(poly)trifluorochloroethylene surfaces. Siloxane bonds or other
appropriate attachment techniques may also be utilized. For
example, siloxane bonds may be formed through reactions of surface
attaching portions which possess trichlorosilyl or trialkoxysilyl
groups. The surface attaching groups additionally possess
functional groups (e.g., amine, hydroxyl, thiol, carboxyl) for
attachment of an oligonucleotide analogue portion. Non-limiting
examples of surface attaching portions include aminoalkylsilanes
and hydroxyalkylsilanes. Non-limiting examples of the surface
attaching portion of the oligonucleotide analog include
bis(2-hydroxyethyl)-amino-propyltriethoxysilane,
aminopropyltriethoxysilane and hydroxypropyl triethoxysilane.
Labeling Compounds
[0060] Many applications in life science research and medical
diagnostics employ one or more types of labels to detect
biomolecules of interest, such as nucleic acids with particular
nucleotide sequences or polypeptides with particular amino acid
sequences. Such applications include many different types of assays
and are used with various technologies, instruments, reagents, etc.
For example, with respect to the analysis of nucleic acids, labels
are utilized within assays which employ microarrays, sequencing,
real-time polymerase chain reaction and many other approaches.
Various assays employ suitable labels of various compositions and
detected through appropriate spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Commonly employed labels include fluorescent, luminescent,
chemiluminescent, light-scattering, and colorimetric varieties.
Labels can be applied directly to a particular target or indirectly
(e.g., through the use of two or more sets of molecules, via an
antibody, via enzymatic labeling systems). Commonly used types of
fluorescent labels include organic dyes (e.g., fluorescein, Cy3,
Cy5, rhodamine), biological fluorophores (e.g.,
phycoerythrocyanin), and quantum dots (e.g., a carboxyl quantum
dot). Fluorescent labels may include, for example,
N-hydroxysuccinimide ester activated dyes that react with exposed
amino groups, malemide activated dyes that react with sulfhydryl
groups, phosphine activated dyes that react with azide groups, or
other suitable labels known in the art. Such labels are available
commercially from, for example, Invitrogen (Carlsbad, Calif.),
Thermo Fisher Scientific (Waltham, Mass.), and ATTO-TEC GmbH
(Siegen, Germany). Antibody labeling techniques include the use of
radiolabels (e.g., removing phosphate groups with an alkaline
phosphatase and replacing the group with a radioactive phosphate
group), enzymatic tags (e.g., horseradish peroxidase), and
fluorescent tags (e.g., utilizing an anti-streptavidin biotinylated
antibody with streptavidin phycoerythrin).
[0061] Assays for the detection of nucleic acids through
hybridization with a microarray often employ one or more labels.
These labels may be directly or indirectly attached, incorporated
or otherwise associated through various means in the art to the
sample nucleic acids. For example, within assays utilizing
amplification during sample preparation, one or more labels may be
incorporated during one or more amplification steps. This may
involve, for instance, the use of polymerase chain reaction (PCR)
with labeled primers and/or nucleotides to provide a labeled
amplification product. Alternative approaches include the addition
of a label to the original nucleic acid sample (e.g., when no
amplification is performed), or to the amplification product. For
example, these approaches may include nick translation labeling to
introduce labeled nucleotides, using a kinase to remove the
phosphate from the end of a nucleic acid fragment and subsequently
incorporate a labeled phosphate (e.g., a radioactive phosphate),
end-labeling using a biotin-labeled deoxynucleotide analog with a
terminal deoxynucleotidyl transferase with subsequent use of
labeled streptavidin, end-labeling through utilization of a ligase
to incorporate a label, or many other approaches known in the art.
Patents with additional non-limiting examples of suitable labels
and methods of using them include U.S. Pat. Nos. 6,864,059;
6,965,020; 7,423,143; 6,864,059; 7,468,243; 7,491,818; 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241, all of which are incorporated herein by reference in
their entirety for all purposes. For a review of methods of
labeling nucleic acids and detecting labeled hybridized nucleic
acids, see Laboratory Techniques in Biochemistry and Molecular
Biology, Vol. 24: Hybridization with Nucleic Acid Probes, P.
Tijssen, ed. Elsevier, N.Y., (1993).
[0062] Means of detecting such labels are well known to those of
skill in the art. For example, radiolabels may be detected using
photographic film or scintillation counters. Fluorescent labels may
be detected using a photodetector to detect fluorescent emissions
after appropriate excitation. Enzymatic labels are typically
detected by providing the enzyme with a substrate and detecting the
reaction product produced by the action of the enzyme on the
substrate, and colorimetric labels are detected by simply
visualizing the colored label.
[0063] Suitable labels may be employed in a "direct" or "indirect"
fashion. Direct labels include those that are directly attached,
bound, incorporated, etc. into the target molecule, such as a
nucleic acid or polypeptide from a sample from an organism of
interest. Thus, as a non-limiting example, if a nucleic acid sample
is to be analyzed through the use of a microarray, a direct
labeling strategy may bind or incorporate labels into the nucleic
acids of the sample before hybridization with the microarray.
[0064] Conversely, indirect labels include those that are attached,
bound, incorporated, etc. into the target molecule in an indirect
fashion, such as through an intermediary molecule. A non-limiting
example well known in the art is to biotinylate nucleic acids
before subsequent use, such as hybridization with a microarray.
Then, the addition of, for example, a streptavidin conjugated
fluorophore, will cause indirect binding of the label to the
nucleic acids of interest for subsequent detection. Of course, many
other label types may be employed, and other biotin binding
techniques may be employed, such as utilizing avidin, streptavidin,
deglycosylated avidin with modified arginines, avidin with a
tyrosine binding site that has been nitrated or iodinated,
anti-biotin antibodies, or other biotin-binding moieties.
Furthermore, continuing with the non-limiting example of
biotinylation, the use of variants and analogs is also possible if
they retain sufficient binding affinity for a particular assay. A
non-limiting example is desthiobiotin, which retains high affinity
with respect to, for instance, streptavidin.
Caged Binding Member Compounds and Methods of Making and Using
[0065] Many approaches are known in the art for caged binding
member compounds and methods of making and using, such as those
which protect a binding member to reduce or eliminate the affinity
the binding member possesses for a particular target binding
species (relative to the affinity the binding member would possess
in its uncaged state for the target binding species). These
approaches can be utilized to prevent binding of a member with
undesired targets which are otherwise capable of binding with the
member, or for other purposes such as controlling the time and
location of binding. Additionally, a variety of approaches can be
utilized to ensure that the affinity of the binding member for the
target binding species is not reduced, at least not substantially,
after uncaging (as compared to the affinity possessed without the
involvement of caging). For instance, a non-limiting example within
the process of manufacturing polymer arrays through
photolithography is the protection of otherwise reactive functional
groups with photolabile protecting groups (e.g., MeNPOC, NNPOC,
NPPOC). These functional groups are then activated for coupling
with monomers within certain regions of the substrate through
selective illumination, with the light possessing wavelength(s)
capable of photolyzing the photolabile protecting groups and
freeing the previously protected, or caged, hydroxyl groups. This
approach of protecting binding members within a cage is certainly
not limited to photolithographic synthesis of nucleic acid arrays,
and many variations and adaptations of the concept are well known
in the art for use with a variety of molecules, such as nucleic
acids, amino acids, antibodies, etc. in a variety of approaches,
chemistries, and applications.
[0066] Certain embodiments herein utilize this concept with respect
to photoprotection of biotin moieties. Specifically, a biotin
molecule (or variant or analog thereof) is modified or otherwise
altered such that it possesses one or more photoactivatable
protecting groups. These protecting groups serve to significantly
reduce the binding affinity that the modified biotin molecule
possesses for avidin (or variants or modified versions thereof,
such as streptavidin) compared to the unmodified state of the
biotin molecule. Some embodiments employ a photoactivatable
protecting group such that appropriate illumination removes the
protecting group to uncage the biotin and restore its natural
binding affinity for the appropriate avidin molecule at issue. As a
non-limiting example, certain embodiments will utilize protective
caging groups that subject to photolysis by illumination in the
ultraviolet spectrum (e.g., illumination containing a wavelength of
365 nm).
[0067] Alternative embodiments employing protected biotin are also
possible. For instance, if avidin is employed to capture a biotin
associated target, such capture can be prevented while the biotin
molecules are still protected within their cages. Selective removal
of the cages to unprotect the biotin at the desired time, location,
etc. allows capture of the biotin associated target by the avidin.
A non-limiting example would be the use of avidin immobilized on a
support to capture biotinylated antibodies, nucleic acids, or
proteins.
[0068] Photoprotection of a molecule, such as biotin, is generally
achieved through modification of the molecule with a
photoactivatable protecting group, with the protecting group
located at a critical position (e.g., deactivating a particular
bond) to prevent undesired reactions while the molecule is still
caged by the protecting group. The inactive, caged molecule is then
uncaged through appropriate irradiation, such as illumination at
one or more appropriate wavelengths. A common example of such
illumination is ultraviolet light. For embodiments where the
protected molecule is associated with molecules that might be
damaged by shorter wavelengths within the ultraviolet spectrum
(e.g., potential damage to DNA by using illumination with
wavelengths shorter than 340 nm), longer wavelengths are more
appropriate (e.g., 350 nm, 360 nm, 365 nm, 375 nm, 390 nm). For
additional background material, see Lusic and Deiters, "A New
Photocaging Group for Aromatic N-Heterocycles," Synthesis, 2006,
No. 13, pp 2147-2150 and Lusic et al., "Photochemical DNA
Activation," Organic Letters, 2007, Vol. 9, No. 10, 1903-1905,
which describe nucleobase caging with 6-nitropiperonyloxymethyl
(NPOM) groups, and which are incorporated herein by reference in
their entireties for all purposes.
[0069] Many approaches are available for the caging of polymers
such as oligonucleotides with photolabile protecting groups. For
example, the caging protecting group may be placed on
internucleotide phosphates, various positions on the sugar, or the
nucleobase. Certain approaches incorporate biotin during
phosphoramidite synthesis of the oligonucleotides. For background
regarding the use of biotin, particularly caged protected biotin,
see U.S. Pat. Nos. 5,252,743; 5,451,683; 6,919,211; and 6,955,915;
U.S. Patent Application Publication No. 2003/0119011; and Pirrung
and Huang, "A General Method for the Spatially Defined
Immobilization of Biomolecules on Glass Surfaces Using "Caged"
Biotin," Bioconjugate Chem., (1996) 7(3): 317-321, all of which are
incorporated herein by reference in their entireties for all
purposes.
[0070] FIGS. 1(A)-1(D) illustrate biotin and protected biotins to
be utilized within certain embodiments. FIG. 1(A) depicts a biotin
100. FIG. 1(B) depicts a biotin 100 with a protecting group 105
(designated as "R") and a cyanoethyl phosphoramidite "CEP" (e.g.,
the nucleoside phosphoramidite as would be utilized in
oligonucleotide synthesis). It should be carefully noted that
although the FIG. 1(B) includes a cyanoethyl phosphoramidite in
association with the depicted protected biotin, many embodiments
herein will not. Instead, these embodiments many employ different
molecules in connection with the valeric acid substituent of the
tetrahydrothiophene ring as may be required or desirable within
certain embodiments. FIGS. 1(B)-1(C) depict examples of the
protecting group 105 from FIG. 1(B). The non-limiting example of a
protective group which is depicted in FIGS. 1(C)-1(D) is
6-nitropiperonyloxymethyl (NPOM). Protected biotin is particularly
useful as in its protected state, its affinity for avidin targets
(e.g., streptavidin) is greatly reduced, but its naturally high
binding affinity will return once appropriate deprotection is
performed.
[0071] As can be seen in FIG. 1(C), a carbamate linkage 110
connects the protecting group 105 of NPOM to the biotin 100, while
FIG. 1(D) depicts an ether linkage 120 which connects the
protecting group 105 of NPOM to biotin 100. The carbamate linkage
110 within FIG. 1(C) is common to many photolabile nitrogen
protecting groups, but possesses disadvantages for many
applications. For example, carbamate linkages are susceptible to
undesired hydrolysis, particularly with respect to aqueous
conditions with an elevated pH, which can lead to unintentional
deprotection of biotin 100 (e.g., hydrolysis which removes the
protecting group that is not mediated by the intended manner of
deprotection, such as application of appropriate photolyzing
illumination). This can make a carbamate linkage disadvantageous
for various applications, including use with amidites (e.g.,
applications involving phosphoramidite chemistry). The ether
linkage 120 in FIG. 1(D), however, resists hydrolysis and thus
prevents undesired deprotection until the desired time and location
where the appropriate illumination is employed. Thus, a biotin 100
as depicted in FIG. 1(B) with a protecting group 105, such as the
non-limiting example of NPOM, utilizing an ether linkage 120 as
illustrated in FIG. 1(D), provides a much more stable
photoprotected biotin that is significantly more likely to only be
deprotected when actually desired and when the appropriate
deprotection means are employed.
[0072] The exact depiction of NPOM with an ether linkage 120, as
illustrated in FIG. 1(D), should not be construed to limit the
embodiments disclosed herein, as not only other photolabile
protecting groups can be used, but also other variants and
modifications of linkages can be employed. For example, FIG. 2
provides additional non-limiting examples of protecting groups 105
and their linkages that can be employed with biotin 100. These
non-limiting examples include linkage variations 210, 220, 230 and
240. Within these variations, X is any suitable substituent,
including but not limited to the non-limiting examples of hydrogen,
alkyl groups, and aryl groups. Many additional variations of these
non-limiting examples will be evident to one of skill in the art
based upon the disclosures herein.
[0073] FIG. 3 depicts non-limiting examples of suitable biotin
molecules 310, 320 and 330 which are protected by one or more
protective groups 105. These examples include, for instance,
positional isomers of the biotin 100 with a protecting group 105
that is depicted in FIG. 1(B). Additionally, as can be observed
within biotin molecule 320, a biotin can be protected by more than
one protecting group 105 to ensure that an extremely low affinity
for avidin, streptavidin, etc. until the protecting groups 105 are
removed. Additionally, appropriate creation of a biotin 320 with
two protecting groups allows the incorporation of two distinct
protecting groups 105 into a particular biotin 320. Thereafter,
careful selection and ordering of deprotecting mechanisms allows
removal of only one protecting group 105 for certain biotins within
an assay to preserve their low affinity for the appropriate biotin
binding protein until such time that the remaining protecting group
105 is also removed.
[0074] FIG. 4 illustrates a comparison of biotin 100 to
desthiobiotin 400. Biotin 100 is depicted with a protecting group
105 (designated as "R"). Desthiobiotin 400 may also possess a
protecting group 405 (designated as "R"), but the R group may also
simply represent H (i.e., desthiobiotin 400 may be utilized in
unprotected forms). Desthiobiotin 400 is an additional non-limiting
example of a biotin variant that also possesses affinity for
avidin, and may be employed in lieu of biotin within many
embodiments and their various applications. Certain embodiments
combine the use of biotin and desthiobiotin, as is discussed
subsequently herein.
[0075] FIG. 5 illustrates a non-limiting synthesis scheme for
producing NPOM protected biotin phosphoramidite. First,
6-nitropiperonyloxymethyl chloride (NPOM-Cl) is coupled with biotin
520. Biotin 520 possesses a protective group 525 (designated as
"R"), and is any suitable protecting group capable of guiding
coupling of NPOM to the tetrahydroimidizalone ring. Subsequently,
the NPOM protected biotin 530 is deprotected to remove protective
group 525. The final step is phosphitylation to produce the NPOM
protected biotin phosphoramidite 540.
[0076] Protected biotin can be employed in a variety of labeling
and detection approaches. In general, these approaches utilize the
protected nature of the biotin prevent binding of avidin (or
streptavidin, etc.) to the biotin with the knowledge that with the
more stable ether linkage that is greatly more resistant to
hydrolysis than, e.g., a more common carbamate linkage, and that
the biotin will be protected from potential avidin binding until
the desired stage of the relevant assay. As discussed previously
herein, biotinylation can occur through a variety of methods known
in the art, including chemical and enzymatic means. Proteins can be
biotinylated, for instance, through primary amine, sulfhydryl,
carboxyl, or glycoprotein biotinylation. Nucleic acids can
incorporate protected biotin through nick translation labeling to
introduce biotin possessing nucleotides, end-labeling using a
biotin-labeled deoxynucleotide analog with a terminal
deoxynucleotidyl transferase, end-labeling through utilization of a
ligase to incorporate a label, or other suitable approaches known
in the art. The biotin is then deprotected at the desirable stage
of the assay, and exposed appropriately for detection suitable for
the assay (e.g., use of an avidin conjugates such as streptavidin
phycoerythrin for subsequent detection of the targets at issue
within the assay.
[0077] Thus, in addition to the use of protected biotin with
non-biotin associated labels, the use of protected biotin can be
combined with the use of unprotected biotin to facilitate a
multi-color approach without the need for distinct binding pairs
for each color. Included within the many embodiments potentially
utilizing protected biotin are microarray assays which utilize a
two color approach. Background on utilizing a multicolor analysis
with microarrays may be found within, for example, Shalon et al.,
"A DNA microarray system for analyzing complex DNA samples using
two-color fluorescent probe hybridization," Genome Res., 6: 639-645
(1996); Yang et al., "Evaluation of experimental designs for
two-color cDNA microarrays," J. Comput. Biol., 12(9): 1202-1220
(2005); Nguyen et al., "Experimental designs for 2-colour cDNA
microarray experiments," Appl. Stochastic Models Bus. Ind., 22:
631-638 (2006); Yatskou et al., "Advanced spot quality analysis in
two-colour microarray experiments, BMC Research Notes, 1: 80
(2008); and Zhu et al., "Assessment of fluorescence resonance
energy transfer for two-color DNA microarray platforms," Anal.
Chem., 82(12): 5304-5312 (2010). Of course, the use of multicolor
analysis is not limited to microarray applications, and embodiments
may be utilized within any suitable application, such as nucleic
acid sequencing (see, e.g., Metzker, "Sequencing technologies--the
next generation," Nature Reviews Genetics, 11: 31-46 (2010));
Ledergerber et al., "Base-calling for next-generation sequencing
platforms," Briefings in Bioinformatics, 12(5): 489-497 (2011); and
Blow, "DNA sequencing: generation next-next," Nature Methods, 5:
267-274 (2008)), or proteomics applications (see, e.g., Waggoner,
"Fluorescent labels for proteomics and genomics," Current Opinion
in Chemical Biology, 10(1): 62-66 (2006); Kleiner et al.,
"Ultra-high sensitivity multi-photon detection imaging in
proteomics analyses," Proteomics, 5(9): 2322-2330 (2005)), and many
other applications known in the art. All of the preceding
references are incorporated herein by reference in their entireties
for all purposes.
[0078] FIG. 6 depicts a non-limiting example of a general labeling
scheme for utilizing multiple types of labels through the use of
biotin with protected biotin that can be employed in many different
applications, as will be apparent to one of skill in the art upon
reading the disclosure herein. Step 1 depicts the results of
biotinylation (through any suitable means, including direct and
indirect techniques) of a first target 610 and a second target 615.
First and second targets 610 and 615 may be any suitable target,
including nucleic acids, proteins, antibodies, cells (e.g.,
biotinylation of cell surface proteins), and any other suitable
target known in the art. Specifically, first target 610 is
biotinylated with an unprotected biotin 100 while second target 615
is biotinylated with a biotin 100 possessing a protecting group
105.
[0079] Step 2 of FIG. 6 depicts the addition of an appropriate
biotin binding protein 620 which is conjugated with a first label
630. Any suitable biotin binding protein can be employed, including
natural, artificial, and modified proteins, which include but are
not limited to avidin, streptavidin, recombinant versions thereof,
ExtrAvidin.RTM. protein (Sigma-Aldrich Corporation, St. Louis,
Mo.), NeutrAvidin.RTM. protein (Thermo Fisher Scientific, Inc.,
Waltham, Mass.), CaptAvidin.TM. protein (Life Technologies
Corporation, Carlsbad, Calif.), and other suitable proteins known
in the art. First label 630 may be any suitable label as discussed
herein and that is desired for the particular labeling scheme at
issue, including the aforementioned non-limiting examples of
fluorescent, luminescent, chemiluminescent, light-scattering, and
colorimetric labels. As seen with Step 2 of FIG. 6, biotin binding
protein 620 binds with the unprotected biotin 100 to effectuate
labeling of first target 610 with first label 630. Meanwhile,
protecting group 105 of the biotin 100 of second target 615 remains
unbound to the biotin binding protein 620 and its conjugated label
630.
[0080] Step 3 of FIG. 6 depicts the deprotection of the biotin 100
of second target 615. The exact manner of deprotection will depend
upon factors such as the particular characteristics of protecting
group 105 (e.g., whether protecting group 105 is photolabile,
acid-labile, base-labile), the relevant assay conditions,
biological and chemical concerns for the various assay components,
and other factors known in the art. Step 3 results in the freeing
of the biotin 100 of second target 615 of protecting group 105, and
thus restoration of the ability for biotin 100 of second target 615
to bind with a suitable biotin binding protein 620. Step 3 may
optionally involve, depending on the assay at issue, washing,
filtering or separation steps to remove the released protecting
group 105.
[0081] Step 4 of FIG. 6 depicts the addition of biotin binding
protein 620, which is conjugated with a second label 635. Second
label 635 is distinguishable from first label 630 under the
appropriate detection conditions (as required by the particular
characteristics of first and second labels 630 and 635). Thus,
second target 615 is labeled with second label 635 through the
binding of the added protein conjugated label. This addition,
however, does not affect the labeling of first target 610 with the
already bound conjugate of biotin binding protein 620 and first
label 630. In many embodiments, the particular biotin binding
protein 620 employed within Step 4 is the same type of protein
utilized within Step 2. In this manner, the same relevant binding
pair of biotin 100 and biotin binding protein 620 is employed for
both first and second targets 610 and 615, ensuring greater
consistency and predictability for the binding scheme in comparison
to the use of binding pairs with different binding affinities.
However, certain assays may utilize a different biotin binding
protein within Step 4 if so desired, because as mentioned above,
many suitable biotin binding proteins are known in the art.
[0082] Furthermore, many variants of the process depicted within
FIG. 6 will be apparent to one of skill in the art. For example,
certain assays may desire to amplify the signal of first and second
labels 630 and 635. This can be achieved, for instance, through the
application in multiple layers of staining, which comprise the
addition of biotinylated antibodies which target biotin binding
protein 620 and the subsequent addition of addition biotin binding
proteins 620 conjugated with the appropriate label. If, for
example, the signal of both first and second labels 630 and 635 are
to be amplified, then such a variation can employ different types
of biotin binding proteins 620 within Steps 2 and 4 respectively.
This can thus ensure, through the use of appropriate antibodies
specific only for the desired biotin binding protein, the proper
amplification of the two labels.
[0083] Moreover, the approach illustrated in FIG. 6 can easily be
expanded to encompass labeling approaches with more than two types
of labels, such as 3, 4, 5, 6, 7, 8, 9, 10 or more types of labels.
Such approaches require the use of different protecting groups 105
for each target which is to be labeled with a different label.
Thus, if a 4 label approach is to be utilized, at least 3 different
protecting groups 105 are employed. By "different" protecting
groups, it is meant that the exact manner of deprotection will be
different. Thus, for the repetitions of Step 3 in the removal of a
particular type of protecting group 105 within a specific assay
step, the deprotection mechanism will only remove that particular
type. Thus, within the example of a 4 label approach, an
unprotected biotin 100 may be employed in combination with: (1) a
biotin 100 protected by a photolabile protecting group 105, (2) a
biotin 100 protected by an acid-labile protecting group 105, and
(3) a biotin 100 protected by a base-labile protecting group. Of
course, not each type of protecting group 105 need be removed
through an entirely different mechanism. For instance, use of
multiple types of photolabile protecting groups is possible if they
are associated with at least partially non-overlapping wavelengths
capable of effectuating photolysis. Relevant factors which guide
selection of the different protecting group include assay concerns
such as the available instruments and reagents, assay condition
boundaries (e.g., pH or salt limitations which must be observed
within various assay stages), and the number of protecting groups.
Additionally, as illustrated within FIG. 3, certain biotins may
encompass two protecting groups 105. Through the use of different
protecting groups 105 for a particular biotin in relation to the
combination of other sets of protecting groups 105 for other
targets within an assay, the use of the same protecting group 105
can be employed in association with multiple labels as long as the
biotin molecules which are not to be bound to a biotin binding
protein 620 in that step maintain their other protecting group
105.
[0084] A non-limiting example of utilizing protected biotin within
a multi-label approach is depicted within FIGS. 7(A)-7(D) and 8. In
this particular illustration, the labeling is performed through the
use of labeling probes which are subsequently incorporated by
ligation. The particular labeling method illustrated here, however,
should not be construed to limit the embodiments disclosed herein,
as any suitable labeling technique known in the art can be suitably
adjusted. FIG. 7(A) depicts a support 700 which possesses a first
oligonucleotide 710 and a second oligonucleotide 720. First
oligonucleotide 710 is designed to hybridize with a portion of
target 715 while second oligonucleotide 720 is designed to
hybridize with a portion of target 725. First oligonucleotide 710
has a reactive end 711 while second oligonucleotide 720 has a
reactive end 721, where reactive end 711 and reactive end 721 are
capable of being ligated to a labeled nucleic acid probe. Within
this particular labeling example which utilizes two labels,
non-hybridized base 716 of target 715 (which is the first base of
target 715 that is not hybridized with first oligonucleotide 710)
can be either an A or T, while the non-hybridized base 726 (which
is the first base of target 725 that is not hybridized with second
oligonucleotide 720) of target 725 can be either a G or C.
[0085] To support 700 is added a first set of labeled nucleic acid
probes 730 and a second set of labeled nucleic acid probes 735. The
first set of labeled probes 730 possesses an A or T at the end of
the probe capable of ligation with reactive end 711, and also
possesses an unprotected biotin 100 (designated as "B"). The
remaining bases of labeled probes 730 may be, for example,
degenerate or universal bases (e.g., 5-nitroindole). The first set
of labeled probes 730 is depicted within FIG. 7(C). The particular
illustration of FIG. 7(C) shows 8 universal bases (designated as
"N"), but this should not be construed to be limiting, as any
suitable number of universal bases may be employed, such as 1-50,
including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 35, 45 or 50,
or an even higher number in certain embodiments. Furthermore,
certain embodiments may not employ any universal bases. The second
set of labeled probes 735 is similar in many aspects to the first
set of labeled probes 730, but with several key distinctions.
First, the base of the probes within the second set 735 which is
capable of ligation to reactive end 721 is either a G or C. Second,
the second set of labeled probes 735 possesses a protected biotin
(designated as "PB"). The second set of labeled probes is further
illustrated within FIG. 7(D), which depicts the 9-mer, biotin 100,
and protecting group 105 (designated as "R"). Protecting group 105
may be, for instance, one of the non-limiting examples provided in
FIG. 2, or any other suitable protecting group as guided by the
requirements or desirable features of the assay at issue.
[0086] FIG. 7(B) depicts the result of utilizing first
oligonucleotide 710 hybridized with first target 715, second
oligonucleotide 720 hybridized with second target 725, the first
set of labeled probes 730 and the second set of labeled probes 735
with an appropriate ligase capable of mismatch discrimination
(e.g., Taq DNA ligase). Based upon the identities of non-hybridized
bases 716 and 726, a probe from the first set of labeled probes 730
has ligated to first oligonucleotide 710 while a probe from the
second set of labeled probes 735 has ligated to second
oligonucleotide 720. This results in first oligonucleotide 710
being labeled with an unprotected biotin while second
oligonucleotide 720 is labeled with a protected biotin.
[0087] FIG. 8 depicts a continuation of the non-limiting example
illustrated within FIGS. 7(A)-7(D). Specifically, FIG. 8 begins
with the depiction of support 700 as illustrated in FIG. 7(B).
Support 700 is shown with first oligonucleotide 710 and second
oligonucleotide 720. While not explicitly shown, within this
particular non-limiting example, first oligonucleotide 710 is still
hybridized with first target 715 and second oligonucleotide 720 is
still hybridized with second target 725. Furthermore, as
illustrated, first oligonucleotide 710 is ligated with a probe from
the first set of labeled probes 730, which contains an unprotected
biotin 100, while second oligonucleotide 720 is ligated with a
probe from the second set of labeled probes 735, which contains a
protected biotin 540. Step 1 of FIG. 7 describes the process
partially illustrated within FIGS. 7(A)-7(B), with the
hybridization of the oligonucleotides to the targets, the ligation
of the labeled probes, and any applicable washing steps (e.g., to
remove unligated labeled probes).
[0088] Step 2 within FIG. 8 illustrates the binding of biotin 100,
which is unprotected, with a suitable biotin binding protein-label
conjugate while protected biotin 540 remains unbound to the biotin
binding protein-label conjugate. The non-limiting example depicted
within Step 2 shows biotin 100 binding with a biotin binding
protein-label conjugate, such as with streptavidin 810 conjugated
with R-phycoerythrin 820. Other labels, including non-fluorescent
labels, are employed in alternative embodiments.
[0089] Step 3 within FIG. 8 illustrates the deprotection of
protected biotin 540. The exact manner of deprotection will depend
upon the protecting group 105 which is employed within a particular
embodiment. For example, if NPOM is employed to protect the biotin,
deprotection can comprise illumination which includes a wavelength
of 365 nm. Other embodiments may utilize photolabile protecting
groups which are photolyzable with different wavelengths, or
protecting groups which are not photolabile (e.g., the use of a
mild acid solution to remove dimethoxytrityl (DMT)). The selection
of an appropriate protecting group and its associated manner of
deprotection is dependent upon many factors, including whether the
deprotection will affect the biotin binding protein-label conjugate
already bound to the biotin which was not protected. Additionally,
the selection is also guided by, for instance, whether the
deprotected biotin will maintain substantially the same binding
affinity in comparison to biotin which was never protected (e.g.,
if the deprotected form retains a structural remnant of the
protection that results in a modified biotin molecule with
decreased affinity with streptavidin). The result of this step
leaves the unprotected biotin 100 with streptavidin 810 and
R-phycoerythrin 820 unaffected, while providing an unprotected
biotin 100 in place of previously protected biotin 540.
[0090] Step 4 within FIG. 8 illustrates the next step, which is the
binding of a distinct avidin-label conjugate, such as streptavidin
810 conjugated with fluorescein 825. As depicted, the streptavidin
810 conjugated with R-phycoerythrin 820 remains unaffected, as only
the biotin 100 which was deprotected within Step 3 is bound within
Step 4 to the streptavidin 810 conjugated with fluorescein 825.
Thus, this process facilitates two-color detection while employing
only one binding interaction through the binding of biotin with
streptavidin. This not only simplified the binding member
interactions at issue within the assay, but also provides a
simplified and more robust multi-color detection scheme that is not
dependent on binding interactions that may involve inferior binding
characteristics. For example, without the protection of biotin, a
two-color assay would require a second binding pair, and this
second binding pair is likely to impose upon the assay the issue of
distinct binding characteristics. Additionally, many binding pairs
will have inferior binding characteristics in comparison to biotin
and streptavidin, and thus will have a relative lower affinity, a
higher off-rate, poorer specificity, etc.
[0091] While the above recited use of protected biotin included the
use of biotin protected by NPOM, a photolabile protecting group, it
should be recognized that other protecting groups, including
non-photolabile protecting groups, may also be employed within
certain embodiments. For example, within FIG. 9, an alternative
version of the protected biotin depicted within FIG. 1(B) is
illustrated, with protecting group 105 comprising dimethoxytrityl
(DMT). Additional non-limiting examples of suitable protecting
groups are also provided within FIG. 9, including azidomethyl
(920), 1-(2-chloroethoxy)ethyl (CEE) (930), 1-(2-cyanoethoxy)ethyl
(940), triisopropylsilyoxymethyl (TIPSOM) (950),
1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP) (960),
1-(2-chloro-4-methylphenyl)-4-methoxypiperidin-4-yl (CTMP) (970)
and allyl (980). Certain embodiments may employ protecting groups
105 which distinct removal conditions so as to facilitate their
combined use within an assay. For example, azidomethyl, illustrated
within FIG. 9 as structure 920, can be removed upon treatment of a
phosphine based reagent (e.g., triphenylphosphine). Further
examples of protecting groups which are suitable for certain
embodiments include those described in "Protecting Groups," Phillip
J. Kocienski, Thieme (3rd Ed. 2005); "Greene's Protecting Groups in
Organic Synthesis," Peter G. M. Wuts and Theodora W. Greene,
Wiley-Interscience (4th Ed. 2006); "Handbook of Synthetic
Photochemistry," Angelo Albini and Maurizio Fagnoni (Eds.),
Wiley-VCH (2010); U.S. Pat. No. 6,147,205 to McGall et al.; U.S.
Pat. No. 7,547,775 to Kuimelis et al.; U.S. Patent Application
Publication No. 2011/0028350; U.S. Patent Application Publication
No. 2003/0040618 to McGall et al.; and U.S. Patent Application
Publication No. 2006/0147969 to Kuimelis et al., all of which are
incorporated herein by reference in their entireties for all
purposes, and particularly for their disclosed protecting groups
and related methods of manufacture and use. As stated earlier,
selection of an appropriate protecting group 105 is based upon
factors such as effective prevention of avidin binding to the
protected biotin at issue, restoration of substantial binding
affinity for the biotin after appropriate deprotection,
compatibility with associated deprotection mechanisms with the
assay (e.g., for the assay depicted in FIG. 9, a deprotection
mechanism should not denature the hybridized nucleic acids through
pH or salt changes), and other factors known in the art.
[0092] DMT is an exemplary non-limiting example of a protecting
group that is not removed through photolysis, but instead through
alternative means, such as treatment with an acid for DMT. Thus,
alternative embodiments of the process illustrated within FIGS.
7(A)-7(D) and 8 utilize a non-photolabile protecting group, such as
DMT, as protecting group 105 for the second set of labeled probes
635. Accordingly, Step 3 within FIG. 8 would be modified such that
deprotection comprises an appropriate means for the non-photolabile
protecting group on protected biotin 540. As a non-limiting
example, if protected biotin 540 was protected by DMT, then Step 3
may comprise treatment with trichloroacetic acid to remove the DMT
and free the biotin for binding with an appropriate avidin-label
conjugate. Other suitable alternative acids may also be employed,
such as trifluoroacetic acid, acetic acid or monopotassium
phosphate. These and other suitable acids may be employed at pH
values of, for example, 2.0, 2.5, 3.0, 3.5, or 4.0. This approach
facilitates use of biotin and the two distinct avidin-label
conjugates as described but without the need for appropriate
illumination to deprotect the biotin by removal of, e.g., the NPOM
protecting group. Such an non-photolabile alternative is helpful in
certain embodiments, such as within those assays where the washing
and staining steps are performed (either manually or in an
automated fashion) without the ability to illuminate the protected
biotin 540 with wavelength(s) appropriate to remove a photolabile
protecting group, or within those assays where the particular
wavelength(s) that would be employed to remove the protecting
groups would damage or otherwise alter one or more components.
[0093] It should be noted that non-limiting example described above
and depicted within FIGS. 7(A)-7(D) and 8 are merely a non-limiting
example of a potential application of the labeling approach more
broadly described herein and depicted within FIG. 6. Regardless of
the particular labeling approach at issue, of which many are known
in the art in addition to the ligation of labeled probes
illustrated in FIGS. 7(A)-7(D) and 8, the basic principles
discussed with respect to FIG. 6 will apply. Thus, it will be
apparent to one of skill in the art to employ such a labeling
approach to many other applications within the life sciences, such
as other nucleic acid and protein array assays, nucleic acid
sequencing, real time PCR, non-array based proteomics applications,
antibody studies, various types of single cell analysis, and other
applications known in the art where labels, including but not
limited to fluorescent labels, are employed.
Biotin Derivatives
[0094] Many embodiments, including the previous embodiments
described herein, may additionally employ biotin derivatives in
place of traditional biotin. One such derivative employed in many
embodiments is desthiobiotin, as was described earlier and
illustrated within FIG. 4. The use of biotin derivatives, such as
desthiobiotin, can be found in, e.g., Hirsch et al., "Easily
reversible desthiobiotin binding to streptavidin, avidin, and other
biotin-binding proteins: uses for protein labeling, detection, and
isolation," Analytical Biochemistry, 308: 343-357 (2002), which is
incorporated herein by reference in its entirety for all purposes.
Desthiobiotin is useful with many embodiments described herein
because biotin possesses a much stronger binding affinity with
streptavidin (1.times.10.sup.15 M.sup.-1 for biotin compared to
5.times.10.sup.13 M.sup.-1 for desthiobiotin) and because
desthiobiotin can be displaced by a biotin solution. This
usefulness is enhanced in view of the ability for biotin binding
with appropriate biotin-binding proteins (e.g., avidin,
streptavidin) to be essentially irreversible under common
conditions for genomic or proteomic assays. For instance, most such
assays are incompatible with common techniques to reverse any
binding that has already occurred through the use of, e.g.,
extremely low pH values or high concentrations of chaotropic
agents. Thus, within most assays, once a biotinylated target (e.g.,
a biotinylated nucleic acid, polypeptide, antibody) is bound with
an appropriate protein (e.g., a streptavidin conjugated with a
fluorophore), the binding and therefore labeling of that target is
permanent for the purposes of the assay. This is one major
contributing factor to the use of distinct binding pairs within
assays, even though such use subjects the associated labeling
process to dependency upon using a binding pair with different
binding affinity values than say, streptavidin with biotin. Such
use of other binding pairs can often lead to less desirable binding
characteristics such as lower affinity, higher off-rate and lower
specificity. Accordingly, these undesirable characteristics are
passed onto the applicable labeling approach for those targets, and
must be appropriately compensated for within subsequent detection
and analysis.
[0095] FIG. 10 depicts a non-limiting example of a general labeling
scheme for the use of multiple types of labels through the use of
biotin with desthiobiotin that can be employed in many different
applications, as will be apparent to one of skill in the art upon
reading the disclosure herein. Step 1 depicts the results of
biotinylation (through any suitable means, including direct and
indirect techniques) of a first target 1010 and a second target
1015. First and second targets 1010 and 1015 may be any suitable
target, including nucleic acids, proteins, antibodies, cells (e.g.,
biotinylation of cell surface proteins), and any other suitable
target known in the art. Specifically, first target 1010 is
biotinylated with an unprotected biotin 100 while second target
1015 is biotinylated with a desthiobiotin 400.
[0096] Step 2 of FIG. 10 depicts the addition of an appropriate
biotin binding protein 1020 which is conjugated with a first label
1030. Any suitable biotin binding protein can be employed,
including natural, artificial, and modified proteins, which include
but are not limited to avidin, streptavidin, recombinant versions
thereof, ExtrAvidin.RTM. protein (Sigma-Aldrich Corporation, St.
Louis, Mo.), NeutrAvidin.RTM. protein (Thermo Fisher Scientific,
Inc., Waltham, Mass.), CaptAvidin.TM. protein (Life Technologies
Corporation, Carlsbad, Calif.), and other suitable proteins known
in the art. First label 1030 may be any suitable label as discussed
herein and that is desired for the particular labeling scheme at
issue, including the aforementioned non-limiting examples of
fluorescent, luminescent, chemiluminescent, light-scattering, and
colorimetric labels. As seen with Step 2 of FIG. 10, biotin binding
protein 1020 binds with both the biotin 100 of first target 1010
and with the desthiobiotin 400 of second target 1015.
[0097] Step 3 of FIG. 10 depicts the displacement of the biotin
binding protein 1020 conjugated with first label 1030 from only
second target 1015. The displacement occurs through the addition of
a biotin solution. As biotin has a significantly greater affinity
for most biotin binding proteins, such as streptavidin, in
comparison to desthiobiotin, the added biotin will essentially
displace the desthiobiotin 400 with respect to the biotin binding
protein 1020 which was previously bound to the desthiobiotin 400 of
second target 1015. The exact manner of biotin displacement will
vary depending on the particular embodiment, the assay conditions,
and many other factors known in the art. For certain embodiments,
use of a 1 mM solution of biotin is sufficient to displace
desthiobiotin 400. However, for other embodiments, the
concentration may need to be adjusted by a person of skill in the
art to suit the specific requirements of the assay at issue. In
most embodiments, Step 3 will also involve the washing, filtering
or otherwise removal of the displaced biotin binding protein 1020
now bound to the newly added biotin 100.
[0098] Step 4 of FIG. 10 depicts the addition of biotin binding
protein 1020, which is conjugated with a second label 1035. Second
label 1035 is distinguishable from first label 1030 under the
appropriate detection conditions (as required by the particular
characteristics of first and second labels 1030 and 1035). Thus,
second target 1015 is labeled with second label 1035 through the
binding of the added protein conjugated label. This addition,
however, does not affect the labeling of first target 1010 with the
already bound conjugate of biotin binding protein 1020 and first
label 1030. In many embodiments, the particular biotin binding
protein 620 employed within Step 4 is the same type of protein
utilized within Step 2. In this manner, the same relevant binding
pair of biotin 100 and biotin binding protein 620 is employed for
both first and second targets 610 and 615. However, the biotin
binding protein 1020 conjugated with second label 1035 may be a
different binding protein if so desired. While the binding
affinities of the two binding pairs are different, the common use
of biotin still provides the advantage of more similar binding
characteristics of the two binding pairs in comparison to many
other alternatives known in the art.
[0099] Furthermore, many variants of the process depicted within
FIG. 10 will be apparent to one of skill in the art. For example,
while the non-limiting example depicted within FIG. 10 employed
desthiobiotin, the principles disclosed herein may be applied with
respect to other suitable biotin analogues and derivatives.
Selection of a suitable biotin analogue or derivative would be
based upon factors such as its binding affinity for the biotin
binding proteins at issue within the assay in relative comparison
to biotin (and relative to any other biotin analogues or
derivatives also employed within the assay). Appropriate selection
of the biotin analogue(s) and/or derivative(s) allows selective
displacement and re-labeling to enable multiple label use,
including 2, 3, 4, 5, 6, 7, 8, 9, 10, and more labels to be
employed.
[0100] Embodiments combining the use of desthiobiotin with biotin
facilitate powerful multiple label approaches. FIGS. 11(A)-11(D)
and 12 illustrate a non-limiting example of such use. The example
depicted within those figures is a modification of the use and
approach described within FIGS. 7(A)-7(D) and 8. Specifically, as
shown in FIG. 11(A), support 1100 possesses a first oligonucleotide
1110 with a reactive end 1111 capable of ligation, and also a
second oligonucleotide 1120 with a reactive end 1121 capable of
ligation. As depicted within FIG. 11(A), first oligonucleotide 1110
has already been hybridized with target 1115 while second
oligonucleotide 1120 has been hybridized with target 1125. The
design of first oligonucleotide 1110 and second oligonucleotide
1120 is such that non-hybridized base 1116 and non-hybridized base
1126 are the first bases of targets 1115 and 1125 which are not
hybridized with the oligonucleotides on the support. Furthermore,
the design has been such that non-hybridized base 1116 is either an
A or T while non-hybridized base 1126 is either a G or C.
[0101] FIG. 11(A) also shows the addition of a first set of labeled
probes 1130 and a second set of labeled probes 1135, which are
illustrated in more detail within FIGS. 11(C) and 11(D)
respectively. Specifically, FIG. 11(A) shows the first set of
labeled probes 1130, which in this particular example possesses 8
universal bases and either an A or T to hybridize with
non-hybridized base 1116, and where "B" represents biotin 100. As
before and as described in association with FIGS. 7(A)-7(D) and 8,
many alterations of this example are possible (e.g., changes to the
number of universal bases). FIG. 11(D) depicts an example of a
probe from the second set of labeled probes 1135, which also has 8
universal bases but instead possesses a G or C to hybridize with
non-hybridized base 1126, and where "DB" represents desthiobiotin
400. Differences between the first set of labeled probes 1130 and
second set of labeled probes 1135 with respect to the first set of
labeled probes 630 and second set of labeled probes 635 is the
absence of protecting groups for the biotin molecule and the
substitution of desthiobiotin within the second set of labeled
probes 1135. Thus, the result of appropriate ligation of the first
and second sets of labeled probes 1130 and 1135 is shown in FIG.
11(B), with first oligonucleotide 1110 ligated to an appropriate
probe of the first set of labeled probes 1130, based on the exact
identity of non-hybridized base 1116, and second oligonucleotide
1120 is ligated to an appropriate probe of the second set of
labeled probes 1135, based on the exact identity of non-hybridized
base 1126.
[0102] The illustration of this particular non-limiting example of
utilizing biotin and desthiobiotin within an assay is continued
within FIG. 12. Specifically, Step 1 within FIG. 12 depicts the
results from FIG. 11(B), with first oligonucleotide 1110 ligated to
a probe of the first set of labeled probes 1130 and second
oligonucleotide 1120 ligated to a probe of the second set of
labeled probes 1135. As depicted in FIGS. 11(A)-11(D), probes from
the first set of labeled probes 1130 possess an unprotected biotin
100 while probes from the second set of labeled probes 1135 possess
desthiobiotin 400. For ease of reference within FIG. 12, targets
1115 and 1125 are not expressly shown, but are, in this particular
non-limiting example, still hybridized with the first and second
oligonucleotides 1110 and 1120 and probes from the first and second
sets of labeled probes 1130 and 1135.
[0103] Step 2 within FIG. 12 depicts the results of binding an
avidin-conjugate, such as streptavidin 1210 conjugated with a first
label 1220. In this particular non-limiting example, the first
label 1220 is a fluorophore, but alternative embodiments employ
other labels, including non-fluorescent labels. Step 2 depicts the
ability of many biotin binding proteins (e.g., avidin,
streptavidin) to bind to both biotin and biotin derivatives (e.g.,
the desthiobiotin employed here), as streptavidin 1210 binds to
both biotin 100 and desthiobiotin 400.
[0104] Step 3 within FIG. 10 show the next step within the assay,
which involves washing with a biotin solution. The biotin within
the solution displaces the desthiobiotin previously bound to
streptavidin 1210 conjugated with the first label 1020. The free
biotin-streptavidin 1210-first label 1220 complex can then be
washed away from support 1100.
[0105] Step 4 within FIG. 10 depicts the next step, where
streptavidin 1210, which is conjugated to a second label 1225
(which is illustrated here as a different fluorescent label than
first label 1220, but which can be any suitable label, including
non-fluorescent labels), is bound to the desthiobiotin 400 of the
second set of labeled probes 1135.
[0106] It should be noted that non-limiting example described above
and depicted within FIGS. 11(A)-11(D) and 12 are merely a
non-limiting example of a potential application of the labeling
approach more broadly described herein and depicted within FIG. 10.
Regardless of the particular labeling approach at issue, of which
many are known in the art in addition to the ligation of labeled
probes illustrated in FIGS. 11(A)-11(D) and 12, the basic
principles discussed with respect to FIG. 10 will apply. Thus, it
will be apparent to one of skill in the art to employ such a
labeling approach to many other applications within the life
sciences, such as other nucleic acid and protein array assays,
nucleic acid sequencing, real time PCR, non-array based proteomics
applications, antibody studies, various types of single cell
analysis, and other applications known in the art where labels,
including but not limited to fluorescent labels, are employed.
Multiple Label Use Combining Biotin, Desthiobiotin, and Protected
Biotin
[0107] As described herein, protected biotin can be employed with
unprotected biotin to facilitate multiple label approaches
(including those utilizing 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
distinct types of labels), where the relevant binding pair (e.g.,
biotin and streptavidin) is the same to facilitate a more effective
and easily analyzed assay that provides superior labeling abilities
in comparison to multiple label approaches which possess different
binding pairs for the labels. Also described herein are approaches
where biotin is employed with desthiobiotin to provide an
alternative avenue for multiple label approaches which take
advantage of the ability of biotin to displace desthiobiotin in its
binding with appropriate biotin binding proteins (e.g., avidin,
streptavidin). However, these approaches can also be combined to
easily facilitate 4 or more label assays which continue to employ
the biotin (and relevant biotin analogues and derivatives) binding
relationship with relevant biotin binding proteins. For example,
the approaches described within FIGS. 6 and 10, directed toward the
use of protected biotin with unprotected biotin and toward the use
of biotin with desthiobiotin, respectively, can additionally be
combined within a labeling scheme. In this manner, four distinct
biotinylated targets can be labeled appropriately with one of four
labels.
[0108] For example, in a combination and expansion of the
approaches described herein with respect to FIGS. 6 and 10, four
different targets of interest can be biotinylated with biotin,
desthiobiotin, biotin protected by a first protecting group, and
biotin protected by a second protecting group, respectively. Then,
through the approaches described herein of adding appropriately
labeled biotin binding proteins conjugated with a label (or
labels), each of the four targets can be appropriately labeled
after deprotection of the protecting group, biotin washing and
displacement of biotin binding proteins bound to the desthiobiotin,
etc. The same concerns will also be relevant in such an application
that is expanded to four (or more labels). For example, with the
use of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 1) protecting
groups within such embodiments, the protecting groups must be
chosen in a manner such that the associated deprotection mechanisms
will not remove other protecting groups in addition to the ones
desirably removed within a specific assay step. For example, within
a four color approach utilizing two different protecting groups, a
non-limiting example may employ DMT to be removed with a
trifluoroacetic acid treatment and azidomethyl to be removed with a
phosphine based treatment.
III. EXAMPLES
Example 1
A Two-Color Coding Method Using DMT Protected Biotin
[0109] An experiment was performed according to the scheme
illustrated within FIGS. 7(A)-7(D) and 8 to demonstrate a two-color
system which utilizes unprotected biotin in combination with DMT
protected biotin. A DNA probe array containing a DNA probe array
containing 5'-phosphorylated 35mer probes was fabricated using
conventional photolithographic techniques. Two synthetic 50mer DNA
targets (10 nM in high-salt buffer) were contacted with the array
for 24 hours at 45.degree. C., with subsequent removal of
non-hybridized target via washing with a reduced salt concentration
buffer for 30 minutes at 37.degree. C. The resulting array was then
contacted with a mixture containing 5'-B--N8-A/T (50 uM),
5'-PB-N8-G/C (50 uM), E. coli DNA ligase (USB Corporation,
Cleveland, Ohio) and E. coli ligase buffer (New England Biolabs,
Inc., NEB, Ipswich, Mass.) for a period of 3 hours at room
temperature. PB is DMT protected biotin, B is unprotected biotin, N
represents degenerate universal bases and A/T or G/C represents a
1:1 ratio of the indicated two bases within the respective probe
set mixtures. Non-ligated components were subsequently removed by
washing the array with low-salt TE buffer at 50.degree. C. Thus,
the initially available biotinylated probes that were not protected
from streptavidin binding were the ones with the A or T as the
9.sup.th base of the probe, and which were ligated to the 5' end of
the 35mer probes. These probes were then stained with
streptavidin-phycoerythrin conjugate by contacting the array with
stain solution for 5-10 minutes before excess reagents were
removed. The next step was to deprotect the biotin of the ligated
G/C probes. The array was treated with a citrate buffer (30 mM, pH
3.5) in NaCl (150 mM) for 30 minutes at room temperature before
excess reagents were removed. The newly available biotinylated
probes (i.e., the probes whose biotin was now deprotected) were
stained by contacting the array with streptavidin-phycoerythrin-Cy5
conjugate, followed by removal of excess reagents.
[0110] Array fluorescence imaging in two separate channels with the
wavelengths appropriate for the two fluorophores reveals the
ability of the protected biotin to resist the first biotin-specific
stain, yet become available for a subsequent biotin-specific stain
step after the biotin protecting group (DMT) was removed with, in
this particular example, a mild acid treatment. The signal obtained
from the fluorescent emissions associated with the
streptavidin-phycoerythrin conjugate bound to the ligated 9mers
with an A or T produced a signal-to-background ratio of 25.9.
Meanwhile, the signal obtained from the fluorescent emissions
associated with the streptavidin-phycoerythrin-Cy5 conjugate bound
to the ligated 9mers with a G or C produced a signal-to-background
ratio of 6.0. Even higher ratios can be obtained with appropriate
selection of the utilized fluorophores.
[0111] FIGS. 13(A)-13(B) contain images of sub-regions of an array
prepared as described above as a demonstration of stability for DMT
protected biotin during a ligation reaction period of 3 hours. The
encircled region within FIG. 13(A) surrounds a set of probes
relevant to the synthetic target 45mer recited above. As can be
seen from a sampling of specific intensity values within FIG. 13(A)
for various positions of the sets of probes, the intensity for
unprotected biotin that is stained with SAPE is extremely strong.
Conversely, within FIG. 13(B), which has an encircled region
surrounding sets of probes ligated with DMT protected biotin after
identical SAPE staining, the sampling of specific intensity values
demonstrates that DMT retains its effectiveness in protecting
biotin from binding with SAPE even after exposure to extended
ligation reactions if the appropriate acid treatment is not
performed to remove the DMT.
[0112] FIGS. 14(A)-14(C) contains demonstrations of the flexibility
of DMT protection for biotin. Specifically, the images within FIG.
14(A) display arrays which underwent detritylation of the DMT
protected biotin with trifluoroacetic acid solutions with pH values
of 2, 3 and 4 for 30 minutes before staining as described above.
FIG. 14(B) contains images corresponding to arrays where the
detritylation of DMT protected biotin occurred via acetic acid
solutions with pH values of 2, 3 and 4 for 30 minutes before
staining FIG. 14(C) contains images corresponding to arrays where
the DMT protected biotin was remove through 20 mM KH.sub.2PO.sub.4
for 30 minutes before staining. The ability of utilizing different
deprotection mechanisms provides flexibility benefits within many
assays, especially those employing orthogonal protection schemes as
appropriate selection of deprotection mechanisms can enhance the
effectiveness and efficiency of utilizing multiple types of biotin
protecting groups to enable additional color capabilities within
multi-color labeling schemes.
Example 2
A Two-Color Coding Method Using Biotin and Desthiobiotin
[0113] An experiment was performed according to the scheme
illustrated within FIGS. 11(A)-11(D) and 12 to demonstrate a
two-color system which utilizes unprotected biotin in combination
with desthiobiotin. A DNA probe array containing a DNA probe array
containing 5'-phosphorylated 35mer probes was fabricated using
conventional photolithographic techniques. Two synthetic 50mer DNA
targets (10 nM in high-salt buffer) were contacted with the array
for 24 hours at 45.degree. C., with subsequent removal of
non-hybridized target via washing with a reduced salt concentration
buffer for 30 minutes at 37.degree. C. The resulting array was then
contacted with a mixture containing 5'-DB--N8-A/T (50 uM),
5'-B-N8-G/C (50 uM), E. coli DNA ligase (USB Corporation,
Cleveland, Ohio) and E. coli ligase buffer (New England Biolabs,
Inc., NEB, Ipswich, Mass.) for a period of 3 hours at room
temperature. DB is desthiobiotin, B is unprotected biotin, N
represents degenerate universal bases and A/T or G/C represents a
1:1 ratio of the indicated two bases within the respective probe
set mixtures. Non-ligated components were subsequently removed by
washing the array with low-salt TE buffer at 50.degree. C. Thus,
the initially available biotinylated probes that were both the ones
with the A or T as the 9.sup.th base of the probe and which
contained desthiobiotin, and also those probes with the G or C as
the 9.sup.th base of the probe and which contained biotin. All
ligated probes were then stained with streptavidin-phycoerythrin
conjugate by contacting the array with stain solution for 5-10
minutes before excess reagents were removed. The next step was to
displace the streptavidin-phycoerythrin conjugate bound to the
desthiobiotin of the ligated A/T probes. This was done by washing
the array with solution biotin (1 mM) in NaCl (150 mM) for 4 hours
at room temperature. The displaced streptavidin-phycoerythrin
conjugate previous bound to the desthiobiotinylated probes was then
removed. The newly available desthiobiotinylated were then stained
by contacting the array with streptavidin-phycoerythrin-Cy5
conjugate, followed by removal of excess reagents.
[0114] Array fluorescence imaging in two separate channels with the
wavelengths appropriate for the two fluorophores reveals the
ability of the unprotected biotin to retain the first stain of
streptavidin-phycoerythrin, and also the ability of the
desthiobiotin to be displaced by a biotin solution and readily bind
to the second stain of streptavidin-phycoerythrin-Cy5 conjugate.
The signal obtained from the fluorescent emissions associated with
the streptavidin-phycoerythrin conjugate bound to the ligated 9mers
with an A or T produced a signal-to-background ratio of 12.5.
Meanwhile, the signal obtained from the fluorescent emissions
associated with the streptavidin-phycoerythrin-Cy5 conjugate bound
to the ligated 9mers with a G or C produced a signal-to-background
ratio of 2.3. Even higher ratios can be obtained with appropriate
selection of the utilized fluorophores.
[0115] FIGS. 15(A)-15(C) contain images of sub-regions with sets of
probes to test the effectiveness of binding and displacement of
desthiobiotin with biotin. Specifically, the probe sets within the
images are ligated with desthiobiotin labeled probes. The image
within FIG. 15(A) shows probes which have been stained with
streptavidin phycoerythrin. The image within FIG. 15(B) shows the
probes after treatment with a 1 mM biotin solution to displace the
desthiobiotin from the SAPE. Washing was also performed to remove
the displaced biotin-SAPE conjugate. FIG. 15(C) contains an image
after subsequent restraining with SAPE. These figures demonstrate
the ability effectively displace and remove a labeling stain from
desthiobiotin, and to effectively re-stain desthiobiotin with a
second biotin binding protein-label conjugate after displacement of
the first biotin binding protein-label conjugate.
[0116] It is to be understood that the above description, including
any examples provided herein, is intended to be illustrative and
not restrictive. Many variations of the invention will be apparent
to those of skill in the art upon reviewing the above description.
The scope of the invention should be determined with reference to
the appended claims, along with the full scope of equivalents to
which such claims are entitled. All cited references, including
patent and non-patent literature, are incorporated herein by
reference in their entirety for all purposes.
* * * * *