U.S. patent application number 12/780446 was filed with the patent office on 2010-12-02 for oligonucleotide paints.
This patent application is currently assigned to President and Fellows of Havard College. Invention is credited to George M. Church, Benjamin Richard Williams, Chao-ting Wu.
Application Number | 20100304994 12/780446 |
Document ID | / |
Family ID | 43220934 |
Filed Date | 2010-12-02 |
United States Patent
Application |
20100304994 |
Kind Code |
A1 |
Wu; Chao-ting ; et
al. |
December 2, 2010 |
Oligonucleotide Paints
Abstract
Novel methods for making high resolution oligonucleotide paints
are provided. Novel, high resolution oligonucleotide paints are
also provided.
Inventors: |
Wu; Chao-ting; (Brookline,
MA) ; Church; George M.; (Brookline, MA) ;
Williams; Benjamin Richard; (Seattle, WA) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
28 STATE STREET, SUITE 1800
BOSTON
MA
02109-1701
US
|
Assignee: |
President and Fellows of Havard
College
Cambridge
MA
|
Family ID: |
43220934 |
Appl. No.: |
12/780446 |
Filed: |
May 14, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61183247 |
Jun 2, 2009 |
|
|
|
61288931 |
Dec 22, 2009 |
|
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Current U.S.
Class: |
506/9 ; 506/16;
506/30 |
Current CPC
Class: |
C12Q 2565/518 20130101;
C12Q 2537/143 20130101; C12Q 1/6841 20130101; C12Q 1/6841
20130101 |
Class at
Publication: |
506/9 ; 506/16;
506/30 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/06 20060101 C40B040/06; C40B 50/14 20060101
C40B050/14 |
Goverment Interests
STATEMENT OF GOVERNMENT INTERESTS
[0002] This invention was made with Government support under the
National Institutes of Health grant number GM085169-01A1. The
Government has certain rights in the invention.
Claims
1. A method of making a set of high resolution oligonucleotide
paints comprising: providing at least one solid support having a
plurality of synthetic, single stranded oligonucleotide sequences
attached thereto, wherein a portion of each of the plurality of
synthetic, single stranded oligonucleotide sequences is
complementary to a portion of a specific chromosome sequence;
synthesizing a plurality of complementary strands, each of which is
complementary to a synthetic, single stranded oligonucleotide
sequence attached to the at least one solid support; removing the
plurality of complementary strands from the at least one solid
support; amplifying the plurality of complementary strands; and
labelling the plurality of complementary strands to produce a set
of high resolution oligonucleotide paints, wherein the set of
oligonucleotides paints has a resolution of about two kilobases or
fewer.
2. The method of claim 1, wherein the set of oligonucleotides
paints has a resolution of about one kilobase or fewer.
3. The method of claim 1, wherein the set of oligonucleotides
paints has a resolution of about 100 bases or fewer.
4. The method of claim 1, wherein the set of oligonucleotides
paints has a resolution of between about 20 bases and about 30
bases.
5. The method of claim 1, wherein 14 bases at each of the 3' and 5'
ends of the oligonucleotide sequences are primer sequences and 32
bases internal to the primer sequences are complementary to a
specific chromosome sequence.
6. The method of claim 1, wherein each of the oligonucleotide
paints has a detectable label attached thereto.
7. The method of claim 1, wherein each of the oligonucleotide
paints has a retrievable label attached thereto.
8. The method of claim 7, wherein the retrievable label further
binds a moiety selected from the group consisting of a protein, a
peptide, a DNA sequence, an RNA sequence and a carbohydrate.
9. The method of claim 8, wherein the retrievable moiety is exposed
to light, heat or a chemical to activate binding of the retrievable
label to a moiety selected from the group consisting of a protein,
a peptide, a DNA sequence, an RNA sequence and a carbohydrate.
10. A method of making a set of oligonucleotide paints comprising:
providing at least one solid support having a plurality of
synthetic, single stranded oligonucleotide sequences attached
thereto, wherein a portion of each of the plurality of synthetic,
single stranded oligonucleotide sequences is complementary to a
portion of a specific chromosome sequence, and wherein each
specific chromosome sequence excludes highly repetitive elements;
synthesizing a plurality of complementary strands, each of which is
complementary to a synthetic, single stranded oligonucleotide
sequence attached to the at least one solid support; removing the
plurality of complementary strands from the at least one solid
support; amplifying the plurality of complementary strands; and
labelling the plurality of complementary strands to produce a set
of oligonucleotide paints.
11. The method of claim 10, wherein each specific chromosome
sequence excludes repetitive elements present in the genome as two
copies, three copies or four copies in a haploid genome.
12. The method of claim 10, wherein the length of each of the
oligonucleotide sequences is about 60 bases.
13. The method of claim 12, wherein 14 bases at each of the 3' and
5' ends of an oligonucleotide sequence are primer sequences and 32
bases internal to the primer sequences are complementary to a
chromosome sequence.
14. The method of claim 10, wherein each of the oligonucleotide
paints has a detectable label attached thereto.
15. The method of claim 10, wherein each of the oligonucleotide
paints has a retrievable label attached thereto.
16. The method of claim 15, wherein the retrievable label further
binds a moiety selected from the group consisting of a protein, a
peptide, a DNA sequence, an RNA sequence and a carbohydrate.
17. The method of claim 15, wherein the retrievable moiety is
exposed to light, heat or a chemical to activate binding of the
retrievable label to a moiety selected from the group consisting of
a protein, a peptide, a DNA sequence, an RNA sequence and a
carbohydrate.
18. The method of claim 14, wherein the detectable label is a
fluorescent label.
19. The method of claim 14, wherein the set of oligonucleotide
paints provides at least 24 spectrally resolvable labels.
20. The method of claim 14, wherein the set of oligonucleotide
paints provides one spectrally resolvable color.
21. The method of claim 14, wherein the set of oligonucleotide
paints provides a spectrally resolvable label for each chromosome
of an organism.
22. The method of claim 14, wherein the set of oligonucleotide
paints provides a spectrally resolvable label for one or more
sub-chromosomal regions of an organism.
23. The method of claim 14, wherein the at least one solid support
is at least one microarray.
24. The method of claim 23, wherein at least 25 microarrays are
provided.
25. The method of claim 23, wherein at least 100 microarrays are
provided.
26. The method of claim 10, wherein the step of amplifying includes
providing a plurality of primers each having a portion that is
complementary to a portion of a complementary strand or a portion
of a single stranded oligonucleotide sequence.
27. The method of claim 26, wherein the primers are universal
primers.
28. The method of claim 26, wherein at least a portion of each of
the primer sequences is removable after said amplification
step.
29. The method of claim 27, wherein the universal primers comprise
between one and 1000 different sequences.
30. The method of claim 27, wherein the universal primers comprise
at least 1000 different sequences.
31. A palette of oligonucleotide paints comprising: a plurality of
oligonucleotide sequences, wherein each oligonucleotide sequence is
complementary to a single type of mutation corresponding to one of
a specific set of chromosome abnormalities associated with a
disorder, and wherein the set comprises at least 50 different
mutations.
32. The palette of oligonucleotide paints of claim 31, wherein the
set comprises at least 100 different types of mutations.
33. The palette of oligonucleotide paints of claim 31, wherein the
set comprises at least 1000 different types of mutations.
34. A set of oligonucleotide paints produced by the method of claim
1.
35. A method of detecting a chromosome rearrangement in a
biological sample comprising: providing a biological sample;
contacting the biological sample with the set of oligonucleotide
paints of claim 34; detecting binding of the set of oligonucleotide
paints; comparing the binding of the set of oligonucleotide paints
to a standard; and detecting a chromosome rearrangement if binding
of the set of oligonucleotide paints differs from the standard.
36. The method of claim 35, wherein the chromosome rearrangement is
selected from the group consisting of translocation, insertion,
inversion, deletion, duplication, transposition, aneuploidy,
polyploidy, complex rearrangement and telomere loss.
37. A method of making a set of oligonucleotide paints comprising:
providing at least one solid support having a plurality of
synthetic, single-stranded oligonucleotide sequences attached
thereto, wherein a portion of each of the plurality of synthetic,
single-stranded oligonucleotide sequences is complementary to a
portion of a specific chromosome sequence; removing the plurality
of synthetic oligonucleotide sequences from the at least one solid
support; amplifying the plurality of synthetic oligonucleotide
sequences to generate amplified, synthetic oligonucleotide
sequences; and labelling the amplified, synthetic oligonucleotide
sequences to produce a set of oligonucleotide paints, wherein each
oligonucleotide paint has a resolution of about two kilobases or
fewer.
38. The method of claim 37, wherein the set of oligonucleotide
paints is fluorescently labelled.
39. The method of claim 37, wherein the set of oligonucleotide
paints provides at least 24 spectrally resolvable labels.
40. The method of claim 37, wherein the set of oligonucleotide
paints provides one spectrally resolvable color.
41. The method of claim 37, wherein the set of oligonucleotide
paints provides a spectrally resolvable label for each chromosome
of an organism.
42. The method of claim 37, wherein the set of oligonucleotide
paints provides a spectrally resolvable label for one or more
sub-chromosomal regions of an organism.
43. The method of claim 37, wherein the at least one solid support
is at least one microarray.
44. The method of claim 43, wherein at least 25 microarrays are
provided.
45. The method of claim 43, wherein at least 100 microarrays are
provided.
46. The method of claim 37, wherein the step of amplifying includes
providing a plurality of primers, each of which is complementary to
a portion of a synthetic oligonucleotide sequence.
47. The method of claim 46, wherein the primers are universal
primers.
48. The method of claim 46, wherein a portion of each of the primer
sequences is removable after said amplification step.
49. A set of oligonucleotide paints produced by the method of claim
37.
50. An article of manufacture for making a set of high resolution
oligonucleotide paints comprising: a plurality of microarrays, each
microarray having a plurality of synthetic oligonucleotide
sequences attached thereto, wherein a portion of each of the
plurality of synthetic oligonucleotide sequences is complementary
to a portion of a specific chromosome sequence, wherein the sum of
synthetic oligonucleotide that are complementary corresponds to
between about 5% and 25% of a genome of interest, and wherein the
set of oligonucleotides paints has a resolution of about two
kilobases or fewer.
51. The article of manufacture of claim 50, wherein the plurality
of synthetic oligonucleotide sequences is complementary to at least
25% of a genome.
52. The article of manufacture of claim 50, further including a
plurality of primers.
53. The article of manufacture of claim 52, wherein the plurality
of primers are universal primers.
54. A kit comprising the set of oligonucleotide paints of claim
37.
55. The kit of claim 54, further comprising instructions for
use.
56. The kit of claim 54, wherein the kit is a diagnostic kit.
57. The kit of claim 56, wherein the kit is used to determine
karyotype of a sample.
58. A kit comprising the set of oligonucleotide paints of claim
49.
59. The kit of claim 58, further comprising instructions for
use.
60. The kit of claim 58, wherein the kit is a diagnostic kit.
61. The kit of claim 60, wherein the kit is used to determine
karyotype of a sample.
62. A method of preparing a plurality of high resolution
oligonucleotide paints comprising: computationally determining
genomic spacing of a plurality of synthetic, oligonucleotide
sequences, wherein each of the plurality is complementary to a
portion of a specific chromosome sequence; synthesizing the
plurality of synthetic oligonucleotide sequences; and labelling the
plurality of synthetic oligonucleotide sequences with a detectable
label to produce a plurality of oligonucleotide paints, wherein the
set of oligonucleotide paints has a resolution of about two
kilobases or fewer, and wherein each of a plurality of the
oligonucleotide paints is complementary to a target nucleic acid
sequence of 40 consecutive nucleotide bases or fewer.
63. The method of claim 62, wherein the plurality of the
oligonucleotide paints is complementary to a target nucleic acid
sequence of 30 consecutive nucleotide bases or fewer.
64. The method of claim 62, wherein the plurality of the
oligonucleotide paints is complementary to a target nucleic acid
sequence of 20 consecutive nucleotide bases or fewer.
65. The method of claim 62, wherein the plurality of the
oligonucleotide paints is complementary to a target nucleic acid
sequence of 10 consecutive nucleotide bases or fewer.
66. The method of claim 62, further comprising the step of
computationally selecting at least one detectable label to label
each of the plurality of synthetic, oligonucleotide sequences.
67. The method of claim 62, further comprising the step of
computationally determining the presence of single nucleotide
polymorphisms in a genomic sequence of interest to reduce synthesis
of synthetic oligonucleotide sequences that bind to repeated
regions of the genomic sequence of interest.
68. A method of making a set of high resolution oligonucleotide
paints comprising: providing at least one solid support having a
plurality of synthetic, single stranded oligonucleotide sequences
attached thereto, wherein a portion of each of the plurality of
synthetic, single stranded oligonucleotide sequences is
complementary to a portion of a specific chromosome sequence;
synthesizing a plurality of complementary strands, each of which is
complementary to a synthetic, single stranded oligonucleotide
sequence attached to the at least one solid support; removing the
plurality of complementary strands from the at least one solid
support; amplifying the plurality of complementary strands; and
labelling the plurality of complementary strands to produce a set
of high resolution oligonucleotide paints, wherein each of a
plurality of the oligonucleotide paints is complementary to a
target nucleic acid sequence of 40 consecutive nucleotide bases or
fewer.
69. The method of claim 68, wherein the plurality of the
oligonucleotide paints is complementary to a target nucleic acid
sequence of 30 consecutive nucleotide bases or fewer.
70. The method of claim 68, wherein the plurality of the
oligonucleotide paints is complementary to a target nucleic acid
sequence of 20 consecutive nucleotide bases or fewer.
71. The method of claim 68, wherein the plurality of the
oligonucleotide paints is complementary to a target nucleic acid
sequence of 10 consecutive nucleotide bases or fewer.
72. The method of claim 68, wherein the plurality of
oligonucleotide paints can cross a cell membrane.
73. The method of claim 68, wherein the plurality of
oligonucleotide paints can cross a nuclear membrane.
74. The method of claim 68, further comprising the step of:
hybridizing the oligonucleotide paints to one or more target
sequences.
75. The method of claim 74, further comprising the step of:
extending the plurality of hybridized oligonucleotide paints.
76. The method of claim 75, wherein the extending step is performed
by primer extension.
77. The method of claim 75, further comprising the step of washing
the extended plurality of hybridized oligonucleotide paints under
stringent conditions.
78. The method of claim 75, wherein the oligonucleotide paints
include a detectable label and a quencher.
79. The method of claim 78, wherein the quencher is released during
the step of extending.
80. The method of claim 78, wherein the detectable label is a
fluorescent label.
81. The method of claim 68, further comprising the step of:
hybridizing the oligonucleotide paints to one or more target
sequences in the presence of an enzyme selected from the group
consisting of one or more of a proteinase, a lipase, and a
ribonuclease.
82. The method of claim 68, wherein the target nucleic acid
sequences are present in a multi-well plate.
83. The method of claim 82, wherein the multi-well plate is a
384-well plate.
84. The method of claim 75, wherein the hybridized oligonucleotide
paints are detected by fluorescent in situ hybridization
(FISH).
85. The method of claim 68, wherein the target nucleic acid
sequence is genomic.
Description
RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
patent application No. 61/183,247, filed Jun. 2, 2009, and
61/228,931, filed Dec. 22, 2009, each of which is hereby
incorporated herein by reference in its entirety for all
purposes.
FIELD
[0003] Embodiments of the present invention relate in general
methods for making and using oligonucleotide paints for chromosome
analysis methods.
BACKGROUND
[0004] Cytogeneticists have been working hand-in-hand with
geneticists and molecular geneticists to clarify the processes of
inheritance and gene expression ever since the synergy of August
Weissman's chromosome theory of inheritance, as interpreted by
Boveri and Sutton in 1902, with Mendel's theory of inheritance, as
brought forth by Morgan, Sturtevant, Muller, and Bridges in their
landmark 1915 publication, The Mechanism of Mendelian Heredity.
This synergy, however, has become technologically unbalanced, as
the tools for dissecting gene expression outstrip those with which
cytogeneticists tease apart the arrangement of chromosomes within
the nucleus or their behavior as they or are inherited from
cell-to-cell or generation-to-generation.
[0005] Chromosome arrangement and behavior cannot be extracted,
purified or captured. Chromosomes have no unit structure that can
be isolated and crystallized, and they produce no product or
enzymatic activity that can be assayed in a test tube. Instead,
researchers must study chromosome arrangement and behavior in situ,
visualizing them with cytological tools or via genetic
manipulation. Constrained as well as guided by these requirements,
remarkable technologies have nonetheless been developed.
Cytology-grade microscopes, electron microscopes, chromosome
stains, and in situ hybridization protocols have all greatly
advanced the ability of scientists to study how chromosome
organization impacts gene expression and development. For example,
the use of fluorescent in situ hybridization (FISH) to reveal the
co-localization of the Myc and Igh genes in transcription
factories, provides a plausible explanation for the frequency with
which these two genes, lying on different chromosomes, become fused
through translocations associated with plasmacytoma and Burkitt
lymphoma (Osborne et al. (2007) PLoS Biol. 5(8):e192). Studies such
as this can only be carried out in situ, highlighting the need for
cytological technologies. Most recently, the technology of
chromosome conformation capture (Ohlsson et al. (2007) Curr. Opin.
Cell Biol. 19(3):321) has fused molecular biological tools and
cytological tools to capture and clone chromosomal regions that
come into contact, generating tremendous excitement among
geneticists and cytogeneticists. Although indirect, genetic
approaches have elucidated the manner by which chromosomes are
transmitted through mitosis and meiosis into subsequent cellular
and organismal generations and, through the use of translocations
and chromosomal rearrangements, demonstrated how chromosome
positioning and interchromosomal interactions can profoundly affect
gene expression (Wu et al. (1999) Curr. Opin. Gen. Dev. 9:237;
Duncan (2002) Ann. Rev. Genet. 36:521; Grant-Downton, et al. (2004)
Trends Genet. 20:188; McKee (2004) Biochim Biophys Acta 1677:165;
Zickler (2006) Chromosoma 115:158).
[0006] Still, scientists remain tremendously limited in the ability
to understand the relationship between chromosome arrangement and
gene expression. Foremost among these needs are technologies that
will permit the visualization of chromosome arrangement, single
nucleus by single nucleus, a need that grows as evidence
accumulates steadily for the roles that chromosome positioning and
interchromosomal interactions play in the regulation of genes and
development in humans and other mammals, Drosophila, plants,
nematodes, fungi and, essentially, every species.
SUMMARY
[0007] Chromosome paints are detectable markers that label
chromosomes along their entire length, permitting physicians and
researchers to identify chromosomes and decipher chromosome
rearrangements. However, commercially available paints are
expensive for routine and frequent use, ranging between $100 to
$4,000 or more per whole genome, per assay, with increased
resolution requiring more expensive paints. As such, many
researchers have not utilized chromosome paints for systematic
genome-wide analysis and have, instead, used the existing
chromosome paint technology sparingly.
[0008] It has been surprisingly discovered that chromosome paints
having superior resolution and labeling functionality can be
economically generated using the methods described herein. It has
been discovered that the per assay cost of chromosome paints could
be reduced approximately 50- to 4,000-fold while increasing
resolution by 100- to 1,000-fold or more, thus rendering possible
many diagnoses and research projects that would otherwise not be
performed or considered due to prohibitive cost. For example, the
methods and compositions described herein can be used to produce
paints for all chromosomes of the human genome for as little as $1
to $2 per assay.
[0009] Accordingly, a first method of making a set of high
resolution oligonucleotide paints is provided. The method includes
the steps of providing at least one solid support having a
plurality of synthetic, single stranded oligonucleotide sequences
attached thereto, wherein a portion of each of the plurality of
synthetic, single stranded oligonucleotide sequences is
complementary to a portion of a specific chromosome sequence,
synthesizing a plurality of complementary strands, each of which is
complementary to a synthetic, single stranded oligonucleotide
sequence attached to the at least one solid support, removing the
plurality of complementary strands from the at least one solid
support, amplifying the plurality of complementary strands, and
labelling the plurality of complementary strands to produce a set
of oligonucleotide paints, wherein the set oligonucleotide paints
has a resolution of about two kilobases or fewer. In certain
aspects, each oligonucleotide paint has a resolution of about one
kilobase or fewer or 100 bases or fewer. In certain aspects, the
set of oligonucleotide paints has a resolution of between about 20
bases and about 30 bases. In certain aspects, the length of each of
the oligonucleotide sequences is about 60 bases (e.g., about 14
bases at each of the 3' and 5' ends of an oligonucleotide sequence
are primer sequences and about 32 bases internal to the primer
sequences are complementary to a chromosome sequence). In other
aspects, each of the oligonucleotide paints has a detectable and/or
retrievable label attached thereto. In certain aspects, the
retrievable label further binds a moiety selected from the group
consisting of a protein, a peptide, a DNA sequence, an RNA sequence
and a carbohydrate. In other aspects, the retrievable moiety is
exposed to light, heat or a chemical to activate binding of the
retrievable label to a moiety selected from the group consisting of
a protein, a peptide, a DNA sequence, an RNA sequence and a
carbohydrate. In certain aspects, each of the oligonucleotide
paints has a detectable label attached thereto. In certain aspects,
the detectable label is a fluorescent label. In other aspects, the
set of oligonucleotide paints provides one spectrally resolvable
color, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, 200, 300, 400, 500
or more spectrally resolvable labels and/or the set of
oligonucleotide paints provides a spectrally resolvable label for
each chromosome and/or one or more sub-chromosomal regions of an
organism. In certain aspects, the plurality of synthetic, single
stranded oligonucleotide sequences encodes 1%, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%, 75%,
80%, 85%, 90%, 95%, 99% or more (e.g., 100%) of a genome (e.g., a
human genome) or between 1% and 75%, between 5% and 50%, between 5%
and 25%, between 5% and 75%, between 10% and 50% or between 20% and
40% of a genome (e.g., a human genome). In certain aspects, at
least 25 microarrays or at least 100 microarrays are provided that
are optionally generated and/or utilized in parallel. In still
other aspects, the step of amplifying includes providing a
plurality of primers (e.g., universal primers), each of which is
complementary to a portion of a complementary strand or a portion
of a single stranded oligonucleotide sequence. In yet other
aspects, at least a portion of each of the primer sequences is
removable after the amplification step. In certain aspects, the
universal primers comprise between one and 1000 different sequences
or comprise at least 1000 different sequences. In other aspects, a
set of oligonucleotide paints produced by the first method is
provided. In still other aspects, a method of detecting a
chromosome rearrangement in a biological sample (e.g., one or more
of translocation, insertion, inversion, deletion, duplication,
transposition, aneuploidy, polyploidy, complex rearrangement and
telomere loss) including the steps of providing a biological
sample, contacting the biological sample with the set of
oligonucleotide paints of the first method, detecting binding of
the set of oligonucleotide paints, comparing the binding of the set
of oligonucleotide paints to a standard, and detecting a chromosome
rearrangement if binding of the set of oligonucleotide paints
differs from the standard is provided.
[0010] A second method of making a set of oligonucleotide paints is
provided. The method includes the steps of providing at least one
solid support having a plurality of synthetic, single stranded
oligonucleotide sequences attached thereto, wherein a portion of
each of the plurality of synthetic, single stranded oligonucleotide
sequences is complementary to a portion of a specific chromosome
sequence and wherein each specific chromosome sequence excludes
highly repetitive elements (and/or any other genomic sequence that
one wants to exclude), synthesizing a plurality of complementary
strands, each of which is complementary to a synthetic, single
stranded oligonucleotide sequence attached to the at least one
solid support, removing the plurality of complementary strands from
the at least one solid support, amplifying the plurality of
complementary strands, and labelling the plurality of complementary
strands to produce a set of oligonucleotide paints. In certain
aspects, each specific chromosome sequence excludes repetitive
elements present in the genome as two copies, three copies or four
copies (i.e., in a haploid genome). In other aspects, the length of
each of the oligonucleotide sequences is about 60 bases (e.g.,
about 14 bases at each of the 3' and 5' ends of an oligonucleotide
sequence are primer sequences and about 32 bases internal to the
primer sequences are complementary to a chromosome sequence). In
certain aspects, each of the oligonucleotide paints has a
retrievable label attached thereto. In certain aspects, the
retrievable label further binds a moiety selected from the group
consisting of a protein, a peptide, a DNA sequence, an RNA sequence
and a carbohydrate. In other aspects, the retrievable moiety is
exposed to light, heat or a chemical to activate binding of the
retrievable label to a moiety selected from the group consisting of
a protein, a peptide, a DNA sequence, an RNA sequence and a
carbohydrate. In certain aspects, each of the oligonucleotide
paints has a detectable label attached thereto. In certain aspects,
the detectable label is a fluorescent label. In other aspects, the
set of oligonucleotide paints provides one spectrally resolvable
color, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, 200, 300, 400, 500
or more spectrally resolvable labels and/or the set of
oligonucleotide paints provides a spectrally resolvable label for
each chromosome and/or one or more sub-chromosomal regions of an
organism. In certain aspects, the plurality of synthetic, single
stranded oligonucleotide sequences encodes 1%, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%, 75% or
more of a genome (e.g., a human genome) or between 1% and 75%,
between 5% and 50%, between 5% and 25%, between 5% and 75%, between
10% and 50% or between 20% and 40% of a genome (e.g., a human
genome). In certain aspects, at least 25 microarrays or at least
100 microarrays are provided that are optionally generated and/or
utilized in parallel. In still other aspects, the step of
amplifying includes providing a plurality of primers (e.g.,
universal primers), each of which is complementary to a portion of
a complementary strand or a portion of a single stranded
oligonucleotide sequence. In yet other aspects, at least a portion
of each of the primer sequences is removable after the
amplification step. In certain aspects, the universal primers
comprise between one and 1000 different sequences or comprise at
least 1000 different sequences. In other aspects, a set of
oligonucleotide paints produced by the second method is provided.
In still other aspects, a method of detecting a chromosome
rearrangement in a biological sample (e.g., one or more of
translocation, insertion, inversion, deletion, duplication,
transposition, aneuploidy, polyploidy, complex rearrangement and
telomere loss) including the steps of providing a biological
sample, contacting the biological sample with the set of
oligonucleotide paints of the second method, detecting binding of
the set of oligonucleotide paints, comparing the binding of the set
of oligonucleotide paints to a standard, and detecting a chromosome
rearrangement if binding of the set of oligonucleotide paints
differs from the standard is provided.
[0011] In certain exemplary embodiments, a palette of
oligonucleotide paints including a plurality of oligonucleotide
sequences, wherein each oligonucleotide sequence is complementary
to a single type of mutation corresponding to one of a specific set
of chromosome abnormalities associated with a disorder, and wherein
the set comprises at least 50 different types of mutations is
provided. In certain aspects, the set includes at least 100
different types of mutations, at least 1000 different types of
mutations, at least 10,000 different types of mutations or
more.
[0012] In certain exemplary embodiments a kit (e.g., a diagnostic
kit) including the set of oligonucleotide paints of the first or
second method is provided. In certain aspects the kit includes
instructions for use. In other aspects, the kit is used to
determine the karyotype of a sample.
[0013] In certain exemplary embodiments, an article of manufacture
for making a set of high resolution oligonucleotide paints is
provided, including a plurality of microarrays, each microarray
having a plurality of synthetic oligonucleotide sequences attached
thereto, wherein a portion of each of the plurality of synthetic
oligonucleotide sequences is complementary to a portion of a
specific chromosome sequence, wherein the sum of synthetic
oligonucleotide that are complementary corresponds to between about
5% and 25% of a genome of interest, and wherein the set of
oligonucleotide paints has a resolution of about two kilobases or
fewer. In other aspects, the plurality of synthetic oligonucleotide
sequences is complementary to approximately 1%, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%,
75% or more of a genome (e.g., a human genome). In still other
aspects, the article of manufacture further includes a plurality of
primers (e.g., universal primers).
[0014] In certain exemplary embodiments, a method of preparing a
plurality of high resolution oligonucleotide paints comprising
computationally determining genomic spacing of a plurality of
synthetic, oligonucleotide sequences, wherein each of the plurality
is complementary to a portion of a specific chromosome sequence,
synthesizing the plurality of synthetic oligonucleotide sequences,
and labelling the plurality of synthetic oligonucleotide sequences
with a detectable label to produce a plurality of oligonucleotide
paints, wherein the set of oligonucleotide paints has a resolution
of about two kilobases or fewer, and wherein each of a plurality of
the oligonucleotide paints is complementary to a target nucleic
acid sequence (e.g., a genomic sequence) of 40 consecutive
nucleotide bases or fewer is provided. In certain aspects, the
plurality of the oligonucleotide paints is complementary to a
target nucleic acid sequence of 30, 20, 10 or fewer consecutive
nucleotide bases. In certain aspects, the method further includes
the step of computationally selecting at least one detectable label
to label each of the plurality of synthetic, oligonucleotide
sequences. In other aspects, the method further includes the step
of computationally determining the presence of single nucleotide
polymorphisms in a genomic sequence of interest to reduce synthesis
of synthetic oligonucleotide sequences that bind to repeated
regions of the genomic sequence of interest.
[0015] In certain exemplary embodiments, a method of making a set
of high resolution oligonucleotide paints comprising providing at
least one solid support having a plurality of synthetic, single
stranded oligonucleotide sequences attached thereto, wherein a
portion of each of the plurality of synthetic, single stranded
oligonucleotide sequences is complementary to a portion of a
specific chromosome sequence, synthesizing a plurality of
complementary strands, each of which is complementary to a
synthetic, single stranded oligonucleotide sequence attached to the
at least one solid support, removing the plurality of complementary
strands from the at least one solid support, amplifying the
plurality of complementary strands, and labelling the plurality of
complementary strands to produce a set of high resolution
oligonucleotide paints, wherein each of a plurality of the
oligonucleotide paints is complementary to a target nucleic acid
sequence of 40 consecutive nucleotide bases or fewer is provided.
In certain aspects, the plurality of the oligonucleotide paints is
complementary to a target nucleic acid sequence of 30, 20, 10 or
fewer consecutive nucleotide bases. In certain aspects, the
oligonucleotide paints can cross a cell membrane and/or a nuclear
membrane. In other aspects, the oligonucleotide paints include a
detectable label (e.g., a fluorescent label) and a quencher. The
quencher can optionally be released during the step of extension.
In certain aspects, the target nucleic acid sequences are present
in a multi-well (e.g., a 384-well) plate. In other aspects,
hybridized oligonucleotide paints are detected by fluorescent in
situ hybridization (FISH). In certain aspects, the target nucleic
acid sequence is genomic.
[0016] In other aspects, a method described herein further includes
the step of hybridizing the oligonucleotide paints to one or more
target sequences. In still other aspects, a method described herein
further includes the step of extending the plurality of hybridized
oligonucleotide paints (e.g., by primer extension). In yet other
aspects, a method described herein includes the step of washing the
extended plurality of hybridized oligonucleotide paints under
stringent conditions. In other aspects, a method described herein
further includes the step of hybridizing the oligonucleotide paints
to one or more target sequences in the presence of an enzyme
selected from the group consisting of one or more of a proteinase,
a lipase, and a ribonuclease.
[0017] Further features and advantages of certain embodiments of
the present invention will become more fully apparent in the
following description of the embodiments and drawings thereof, and
from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee. The foregoing and
other features and advantages of the present invention will be more
fully understood from the following detailed description of
illustrative embodiments taken in conjunction with the accompanying
drawings in which:
[0019] FIG. 1 schematically depicts one method of making of
chromosome paints (i.e., Oligopaints) from oligonucleotides.
[0020] FIG. 2 schematically depicts one method of making 20% of the
human genome on up to 92 arrays which, after amplification with
between 5 and 15 (=5+1+1+1+7) different kinds of primer pairs, will
generate 664 pools of genomic sequence. The pools can be combined
in a variety of ways to target chromosomes or sub-chromosomal
regions.
[0021] FIG. 3 schematically depicts how thick banding will become a
finer pattern on decondensed chromosomes.
[0022] FIG. 4 schematically depicts strategies for aliquoting.
Either strategy allows for the visualization of just one or a few
chromosomes at a time, as well as permitting the visualization of
sub-chromosomal regions. Aliquots carrying different primer
sequences can be labeled with the same marker if there is no need
to distinguish the targets by fluorescent in situ hybridization
(FISH).
[0023] FIG. 5 schematically depicts one protocol to make Oligopaint
probes from chip-synthesized oligonucleotide libraries.
[0024] FIG. 6 depicts an RNAi screen for genes involved in
Drosophila cells, locked nucleic acid (LNA) probes and automated
scoring.
[0025] FIG. 7 schematically depicts the use of PCR primers that
include an internal dU and an internal fluor, enabling digestion of
the 5' end of the primers with USER.TM. (uracil-specific excision
reagent) (New England Biolabs, Ipswich, Mass.).
DETAILED DESCRIPTION
[0026] The principles of the present invention may be applied with
particular advantage in methods of tagging (i.e., painting with
chromosome paints) one or more oligonucleotide sequences, e.g.,
chromosome regions (e.g., sub-chromosomal regions) and/or one or
more entire chromosomes. The methods described herein create
chromosome paints that have an increased resolution over
commercially available chromosome paints, which is due in part to
the fact that the chromosome paints are synthesized using a
specific set of primers, which can amplify and label specific
sequences with near absolute certainty. Thus, the chromosome paints
described herein have a theoretical resolution on the order of base
pairs.
[0027] Exemplary embodiments of the present invention are directed
to methods for generating novel chromosome paints using synthetic
genomic template sequences (e.g., genomic template sequences that
have been synthesized on arrays). The synthetic genomic template
sequences can be, for example, synthetic genomic template sequences
that are generated on and subsequently released from an array into
one or more pools, or extension products which are made using
synthetic genomic template sequences attached to an array as a
template and then released into one or more pools by melting. The
released sequences are then amplified and labeled to produce
chromosome paints. By designing synthetic genomic template
sequences to be flanked by primer sequences, the primers can be
used both to label the synthetic genomic template sequences as well
as to amplify the genomic sequence. Labeling a chromosome paint can
be performed by a variety of methods including, but not limited to,
using primers that have been pre-labeled, incorporating labels
during amplification or indirect labeling. Labels and methods of
incorporating labels into oligonucleotide sequences are discussed
further herein.
[0028] As used herein, the term "chromosome paint" refers to
detectably labeled polynucleotides that have sequences
complementary to DNA sequences from a particular chromosome or
sub-chromosomal region of a particular chromosome. Chromosome
paints that are commercially available are derived from
fluorescence activated cell sorted (FACS) and/or flow sorted
chromosomes or from bacterial artificial chromosomes (BACs) or
yeast artificial chromosomes (YACs). As such, chromosome paints
known in the art at the time of filing were laborious to generate
and are limited in their resolution.
[0029] As used herein, the term "Oligopaint" refers to detectably
labeled polynucleotides that have sequences complementary to an
oligonucleotide sequence, e.g., a portion of a DNA sequence e.g., a
particular chromosome or sub-chromosomal region of a particular
chromosome. Oligopaints are generated from synthetic probes and
arrays that are, optionally, computationally patterned (rather than
using natural DNA sequences and/or chromosomes as a template).
[0030] Since Oligopaints are generated using nucleic acid sequences
that are present in a pool, they are no longer spatially
addressable (i.e., no longer attached to an array). Surprisingly,
however, this method increases resolution of the chromosome paints
over those that are made using yeast artificial chromosomes (YACs),
bacterial artificial chromosomes (BACs), and/or flow sorted
chromosomes. In certain aspects, the Oligopaints described herein
have a resolution that is, e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,
125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 350%, 400%, 450%,
500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%,
6000%, 7000%, 8000%, 9000%, 10,000%, 100,000%, 1,000,000%,
10,000,000%, 100,000,000% or greater than chromosome paints that
are commercially available.
[0031] Typically, chromosome paints that are commercially available
have a chromosome resolution on the order of at least
6.times.10.sup.6 base pairs. The Oligopaints described herein,
however, have a much higher resolution when compared with paints
known in the art. As used herein, the term "resolution" refers to
the ability to distinguish (e.g., label) between two points on a
polynucleotide sequence (e.g., two points along the length of a
chromosome). As used herein, the term "high resolution" refers to
the ability to detect two or more nucleic acid sequences having a
distance of less than 6.times.10.sup.6 base pairs apart (e.g., on a
chromosome). In certain aspects, two or more high resolution
Oligopaints have a resolution of about 500 kilobases apart or
fewer, 400 kilobases apart or fewer, 300 kilobases apart or fewer,
200 kilobases apart or fewer, 100 kilobases apart or fewer, 90
kilobases apart or fewer, 80 kilobases apart or fewer, 70 kilobases
apart or fewer, 60 kilobases apart or fewer, 50 kilobases apart or
fewer, 40 kilobases apart or fewer, 30 kilobases apart or fewer, 20
kilobases apart or fewer, 19 kilobases apart or fewer, 18 kilobases
apart or fewer, 17 kilobases apart or fewer, 16 kilobases apart or
fewer, 15 kilobases apart or fewer, 14 kilobases apart or fewer, 13
kilobases apart or fewer, 12 kilobases apart or fewer, 11 kilobases
apart or fewer, 10 kilobases apart or fewer, 9 kilobases apart or
fewer, 8 kilobases apart or fewer, 7 kilobases apart or fewer, 6
kilobases apart or fewer, 5 kilobases apart or fewer, 4 kilobases
apart or fewer, 3 kilobases apart or fewer, 2 kilobases apart or
fewer or 1 kilobase apart or fewer. In certain aspects, two or more
high resolution Oligopaints have a resolution of about 1900 bases
apart or fewer, 1800 bases apart or fewer, 1700 bases apart or
fewer, 1600 bases apart or fewer, 1500 bases apart or fewer, 1400
bases apart or fewer, 1300 bases apart or fewer, 1200 bases apart
or fewer, 1100 bases apart or fewer, 1000 bases apart or fewer, 900
bases apart or fewer, 800 bases apart or fewer, 700 bases apart or
fewer, 600 bases apart or fewer, 500 bases apart or fewer, 400
bases apart or fewer, 300 bases apart or fewer, 200 bases apart or
fewer, 100 bases apart or fewer, 95 bases apart or fewer, 90 bases
apart or fewer, 85 bases apart or fewer, 80 bases apart or fewer,
75 bases apart or fewer, 70 bases apart or fewer, 65 bases apart or
fewer, 60 bases apart or fewer, 55 bases apart or fewer, 50 bases
apart or fewer, 45 bases apart or fewer, 40 bases apart or fewer,
35 bases apart or fewer, 30 bases apart or fewer, 25 bases apart or
fewer, 20 bases apart or fewer, 15 bases apart or fewer, 10 bases
apart or fewer or down to the individual base pair. In certain
aspects, two or more high resolution Oligopaints have a resolution
of between about 10 bases and about 2000 bases, between about 10
bases and about 1000 bases, between about 10 bases and about 500
bases, between about 15 bases and about 250 bases, between about 15
bases and about 100 bases, between about 20 bases and about 50
bases, or between about 20 bases and about 30 bases.
[0032] The sensitivity of resolution of Oligopaints described
herein is much greater than paints known in the art. As used
herein, the term "sensitivity," with respect to Oligopaints, refers
to the number of target nucleotide bases (e.g., target genomic
nucleotide bases) that are complementary to a particular
Oligopaint, i.e., the number of target nucleotide bases to which a
particular Oligopaint can hybridize (i.e., the smallest band size
that can be detected). In certain aspects, high resolution
Oligopaints have a resolution of about 1 kilobase, about 1900
bases, about 1800 bases, about 1700 bases, about 1600 bases apart,
about 1500 bases, about 1400 bases, about 1300 bases, about 1200
bases, about 1100 bases, about 1000 bases, about 900 bases, about
800 bases, about 700 bases, about 600 bases, about 500 bases, about
400 bases, about 300 bases, about 200 bases, about 100 bases, about
95 bases, about 90 bases, about 85 bases, about 80 bases, about 75
bases, about 70 bases, about 65 bases, about 60 bases, about 55
bases, about 50 bases, about 45 bases, about 40 bases, about 35
bases, about 30 bases, about 25 bases, about 20 bases, about 15
bases, about 10 bases, or about 5 bases. In certain aspects, the
number of target nucleotide bases that are complementary to an
Oligopaint are consecutive (e.g., consecutive genomic nucleotide
bases).
[0033] In certain exemplary embodiments, Oligopaints are
complementary to genomic nucleic sequences that are present in low
or single copy numbers (e.g., genomic nucleic sequences that are
not repetitive elements). As used herein, the term "repetitive
element" refers to a DNA sequence that is present in many identical
or similar copies in the genome. Repetitive elements are not
intended to refer to a DNA sequence that is present on each copy of
the same chromosome (e.g., a DNA sequence that is present only
once, but is found on both copies of chromosome 11, would not be
considered a repetitive element, and would be considered a sequence
that is present in the genome as one copy). The genome consists of
three broad sequence components: Single copy or at least very low
copy number DNA (approximately 60% of the human genome); moderately
repetitive elements (approximately 30% of the human genome); and
highly repetitive elements (approximately 10% of the human genome).
For a review, see Human Molecular Genetics, Chapter 7 (1999), John
Wiley & Sons, Inc.
[0034] In certain exemplary embodiments, small Oligopaints are
provided. As used herein, the term "small Oligopaint" refers to an
Oligopaint of between about 5 bases and about 100 bases long, or an
Oligopaint of about 5 bases, about 10 bases, about 15 bases, about
20 bases, about 25 bases, about 30 bases, about 35 bases, about 40
bases, about 45 bases, about 50 bases, about 55 bases, about 60
bases, about 65 bases, about 70 bases, about 75 bases, about 80
bases, about 85 bases, about 90 bases, about 95 bases, or about 100
bases. Small Oligopaints can access targets that are not accessible
to longer oligonucleotide probes. For example, in certain aspects
small Oligopaints can pass into a cell, can pass into a nucleus,
and/or can hybridize with targets that are partially bound by one
or more proteins, etc. Small Oligopaints are also useful for
reducing background, as they can be more easily washed away than
larger hybridized oligonucleotide sequences.
[0035] In certain exemplary embodiments, the length of an
Oligopaint can be increased (e.g., by primer extension) after it
has been hybridized to a target sequence, e.g., a target genomic
sequence. Such an extension can increase the binding affinity of
the Oligopaint to the target sequence, allowing more stringent
hybridization and/or wash conditions to be used (temperature, salt
concentration, detergent concentration and the like, discussed
further herein) as compared to a shorter Oligopaint while still
allowing the use of small Oligopaints. In certain aspects, the use
of stringent hybridization and/or wash conditions improves the
signal to noise ratio of an Oligopaint.
[0036] As used herein, the terms "Oligopainted" and "Oligopainted
region" refer to a target nucleotide sequence (e.g., a chromosome)
or region of a target nucleotide sequence (e.g., a sub-chromosomal
region), respectively, that has hybridized thereto one or more
Oligopaints. Oligopaints can be used to label a target nucleotide
sequence, e.g., chromosomes and sub-chromosomal regions of
chromosomes during various phases of the cell cycle including, but
not limited to, interphase, preprophase, prophase, prometaphase,
metaphase, anaphase, telophase and cytokenesis.
[0037] As used herein, the term "chromosome" refers to the support
for the genes carrying heredity in a living cell, including DNA,
protein, RNA and other associated factors. The conventional
international system for identifying and numbering the chromosomes
of the human genome is used herein. The size of an individual
chromosome may vary within a multi-chromosomal genome and from one
genome to another. A chromosome can be obtained from any species. A
chromosome can be obtained from an adult subject, a juvenile
subject, an infant subject, from an unborn subject (e.g., from a
fetus, e.g., via prenatal test such as amniocentesis, chorionic
villus sampling, and the like or directly from the fetus, e.g.,
during a fetal surgery) from a biological sample (e.g., a
biological tissue, fluid or cells (e.g., sputum, blood, blood
cells, tissue or fine needle biopsy samples, urine, cerebrospinal
fluid, peritoneal fluid, and pleural fluid, or cells therefrom) or
from a cell culture sample (e.g., primary cells, immortalized
cells, partially immortalized cells or the like). In certain
exemplary embodiments, one or more chromosomes can be obtained from
one or more genera including, but not limited to, Homo, Drosophila,
Caenorhabiditis, Danio, Cyprinus, Equus, Canis, Ovis, Ocorynchus,
Salmo, Bos, Sus, Gallus, Solanum, Triticum, Oryza, Zea, Hordeum,
Musa, Avena, Populus, Brassica, Saccharum and the like.
[0038] As used herein, the term "chromosome banding" refers to
differential staining of chromosomes resulting in a pattern of
transverse bands of distinguishable (e.g., differently or
alternately colored) regions, that is characteristic for the
individual chromosome or chromosome region (i.e., the "banding
pattern"). Conventional banding techniques include G-banding
(Giemsa stain), Q-banding (Quinacrine mustard stain), R-banding
(reverse-Giemsa), and C-banding (centromere banding).
[0039] As used herein, the term "karyotype" refers to the
chromosome characteristics of an individual cell, cell line or
genome of a given species, as defined by both the number and
morphology of the chromosomes. Karyotype can refer to a variety of
chromosomal rearrangements including, but not limited to,
translocations, insertional translocations, inversions, deletions,
duplications, transpositions, anueploidies, complex rearrangements,
telomere loss and the like. Typically, the karyotype is presented
as a systematized array of prophase or metaphase (or otherwise
condensed) chromosomes from a photomicrograph or computer-generated
image. Interphase chromosomes may also be examined.
[0040] As used herein, the terms "chromosomal aberration" or
"chromosome abnormality" refer to a deviation between the structure
of the subject chromosome or karyotype and a normal (i.e.,
non-aberrant) homologous chromosome or karyotype. The deviation may
be of a single base pair or of many base pairs. The terms "normal"
or "non-aberrant," when referring to chromosomes or karyotypes,
refer to the karyotype or banding pattern found in healthy
individuals of a particular species and gender. Chromosome
abnormalities can be numerical or structural in nature, and
include, but are not limited to, aneuploidy, polyploidy, inversion,
translocation, deletion, duplication and the like. Chromosome
abnormalities may be correlated with the presence of a pathological
condition or with a predisposition to developing a pathological
condition. Chromosome aberrations and/or abnormalities can also
refer to changes that are not associated with a disease, disorder
and/or a phenotypic change. Such aberrations and/or abnormalities
can be rare or present at a low frequency (e.g., a few percent of
the population (e.g., polymorphic)).
[0041] Disorders associated with one or more chromosome
abnormalities include, but are not limited to: autosomal
abnormalities (e.g., trisomies (Down syndrome (chromosome 21),
Edwards syndrome (chromosome 18), Patau syndrome (chromosome 13),
trisomy 9, Warkany syndrome (chromosome 8), trisomy 22/cat eye
syndrome, trisomy 16); monosomies and/or deletions (Wolf-Hirschhorn
syndrome (chromosome 4), Cri du chat/Chromosome 5q deletion
syndrome (chromosome 5), Williams syndrome (chromosome 7), Jacobsen
syndrome (chromosome 11), Miller-Dieker syndrome/Smith-Magenis
syndrome (chromosome 17), Di George's syndrome (chromosome 22),
genomic imprinting (Angelman syndrome/Prader-Willi syndrome
(chromosome 15))); X/Y-linked abnormalities (e.g., monosomies
(Turner syndrome (XO), trisomy or tetrasomy and/or other karyotypes
or mosaics (Klinefelter's syndrome (47 (XXY)), 48 (XXYY), 48 (XXXY)
49 (XXXYY), 49 (XXXXY), Triple X syndrome (47 (XXX)), 48 (XXXX), 49
(XXXXX) 47 (XYY), 48 (XYYY), 49 (XYYYY), 46 (XX/XY));
translocations (e.g., leukemia or lymphoma (e.g., lymphoid (e.g.,
Burkitt's lymphoma t(8 MYC; 14 IGH) , follicular lymphoma t(14 IGH;
18 BCL2), mantle cell lymphoma/multiple myeloma t(11 CCND1; 14
IGH), anaplastic large cell lymphoma t(2 ALK; 5 NPM1), acute
lymphoblastic leukemia) or myeloid (e.g., Philadelphia chromosome
t(9 ABL; 22 BCR), acute myeloblastic leukemia with maturation t(8
RUNX1T1;21 RUNX1), acute promyelocytic leukemia t(15 PML,17 RARA),
acute megakaryoblastic leukemia t(1 RBM15;22 MKL1))) or other
(e.g., Ewing's sarcoma t(11 FLI1; 22 EWS), synovial sarcoma t(x
SYT;18 SSX), dermatofibrosarcoma protuberans t(17 COL1A1; 22
PDGFB), myxoid liposarcoma t(12 DDIT3; 16 FUS), desmoplastic small
round cell tumor t(11 WT1; 22 EWS), alveolar rhabdomyosarcoma t(2
PAX3; 13 FOXO1) t (1 PAX7; 13 FOXO1))); gonadal dysgenesis (e.g.,
mixed gonadal dysgenesis, XX gonadal dysgenesis); and other
abnormalities (e.g., fragile X syndrome, uniparental disomy).
Disorders associated with one or more chromosome abnormalities also
include, but are not limited to, Beckwith-Wiedmann syndrome,
branchio-oto-renal syndrome, Cri-du-Chat syndrome, De Lange
syndrome, holoprosencephaly, Rubinstein-Taybi syndrome and WAGR
syndrome.
[0042] Disorders associated with one or more chromosome
abnormalities also include cellular proliferative disorders (e.g.,
cancer). As used herein, the term "cellular proliferative disorder"
includes disorders characterized by undesirable or inappropriate
proliferation of one or more subset(s) of cells in a multicellular
organism. The term "cancer" refers to various types of malignant
neoplasms, most of which can invade surrounding tissues, and may
metastasize to different sites (see, for example, PDR Medical
Dictionary 1st edition, 1995). The terms "neoplasm" and "tumor"
refer to an abnormal tissue that grows by cellular proliferation
more rapidly than normal and continues to grow after the stimuli
that initiated proliferation is removed (see, for example, PDR
Medical Dictionary 1st edition, 1995). Such abnormal tissue shows
partial or complete lack of structural organization and functional
coordination with the normal tissue which may be either benign
(i.e., benign tumor) or malignant (i.e., malignant tumor).
[0043] Disorders associated with one or more chromosome
abnormalities also include brain disorders including, but not
limited to, acoustic neuroma, acquired brain injury, Alzheimer's
disease, amyotrophic lateral diseases, aneurism, aphasia,
arteriovenous malformation, attention deficit hyperactivity
disorder, autism Batten disease, Bechet's disease, blepharospasm,
brain tumor, cerebral palsy Charcot-Marie-Tooth disease, chiari
malformation, CIDP, non-Alzheimer-type dementia, dysautonomia,
dyslexia, dysprazia, dystonia, epilepsy, essential tremor,
Friedrich's ataxia, gaucher disease, Gullian-Barre syndrome,
headache, migraine, Huntington's disease, hydrocephalus, Meniere's
disease, motor neuron disease, multiple sclerosis, muscular
dystrophy, myasthenia gravis, narcolepsy, Parkinson's disease,
peripheral neuropathy, progressive supranuclear palsy, restless
legs syndrome, Rett syndrome, schizophrenia, Shy Drager syndrome,
stroke, subarachnoid hemorrhage, Sydenham's syndrome, Tay-Sachs
disease, Tourett syndrome, transient ischemic attack, transverse
myelitis, trigeminal neuralgia, tuberous sclerosis and von
Hippel-Lindau syndrome.
[0044] In certain exemplary embodiments, Oligopaint kits are
provided. As used herein, the term "kit" refers to any delivery
system for delivering Oligopaints and/or reagents for carrying out
a method described herein. In the context of assays, such kits
include systems that allow for the storage, transport, or delivery
of reaction reagents (e.g., an enclosure providing one or more of,
e.g., Oligopaints, primers (e.g., primers specific for all
Oligopaints present and/or one or more subsets of primers specific
to one or more subsets of Oligopaint sequences) primers having one
or more detectable and/or retrievable labels bound thereto),
supports having oligonucleotides bound thereto (e.g., microarrays,
palettes, etc.), or the like) and/or supporting materials (e.g., an
enclosure providing, e.g., buffers, written instructions for
performing an assay described herein, or the like) from one
location to another. For example, kits include one or more
enclosures (e.g., boxes) containing the relevant reaction reagents
and/or supporting materials for assays described herein. In one
aspect, kits of the invention comprise Oligopaints specific for one
or more target nucleotide sequences (e.g., chromosomes) or one or
more regions of one or more target nucleotide sequences (e.g.,
sub-chromosomal regions). In another aspect, kits comprise one or
more primer sequences, one or more supports having a plurality of
synthetic, oligonucleotide sequences attached thereto, and one or
more detectable and/or retrievable labels. Such contents may be
delivered to the intended recipient together or separately. For
example, a first container may contain primer sequences for use in
an assay, while a second container may contain a support having a
plurality of synthetic, oligonucleotide sequences attached
thereto.
[0045] In certain embodiments, an Oligopaint kit provides one or
more arrays and/or palettes having a plurality of specific
oligonucleotide sequences (e.g., Oligopaints) bound thereto. In
certain aspects, an array and/or palette provides a plurality of
oligonucleotide sequences (e.g., Oligopaints) that is specific for
a set of binding patterns in a genome (e.g., a human genome). In
certain aspects, an array or palette is specific for a set of
chromosomal aberrations (e.g., one or more of a translocation, an
insertion, an inversion, a deletion, a duplication, a
transposition, aneuploidy, polyploidy, complex rearrangement and
telomere loss) associated with one or more disorders described
herein. In certain aspects, the Oligopaint kits described herein
are particularly suited for diagnostic and/or prognostic use for
detecting one or more disorders described herein in clinical
settings (e.g., hospitals, medical clinics, medical offices,
diagnostic laboratories, research laboratories and the like (e.g.,
for patient diagnosis and/or prognosis, prenatal diagnosis and/or
prognosis and the like).
[0046] In certain aspects, an Oligopaint kit provides instructions
for amplifying the plurality of specific oligonucleotide sequences
(e.g., Oligopaints) provided in the kit. In other aspects, the kit
provides instructions for detectably and/or retrievably labeling
one or more target nucleic acid sequences (e.g., one or more
chromosomes or sub-chromosomal regions) using the amplified
Oligopaints. In other aspects, an Oligopaint kit provides
instructions for effectively removing one or more of the plurality
of specific oligonucleotide sequences (e.g., Oligopaints) during
the amplification step by including one or more unlabeled
amplification primers that hybridizes to the one or more
oligonucleotide sequences that one wishes to remove, such that the
one or more target nucleic acid sequences is rendered not
detectably and/or retrievably labeled.
[0047] In certain exemplary embodiments, a polynucleotide (e.g., an
Oligopaint) has a retrievable label bound thereto. As used herein,
the terms "bound" and "attached" refer to both covalent
interactions and noncovalent interactions. A covalent interaction
is a chemical linkage between two atoms or radicals formed by the
sharing of a pair of electrons (i.e., a single bond), two pairs of
electrons (i.e., a double bond) or three pairs of electrons (i.e.,
a triple bond). Covalent interactions are also known in the art as
electron pair interactions or electron pair bonds. Noncovalent
interactions include, but are not limited to, van der Waals
interactions, hydrogen bonds, weak chemical bonds (i.e., via
short-range noncovalent forces), hydrophobic interactions, ionic
bonds and the like. A review of noncovalent interactions can be
found in Alberts et al., in Molecular Biology of the Cell, 3d
edition, Garland Publishing, 1994.
[0048] As used herein, the term "retrievable label" refers to a
label that is attached to a polynucleotide (e.g., an Oligopaint)
and can, optionally, be used to specifically and/or nonspecifically
bind a target protein, peptide, DNA sequence, RNA sequence,
carbohydrate or the like at or near the nucleotide sequence to
which one or more Oligopaints have hybridized. In certain aspects,
target proteins include, but are not limited to, proteins that are
involved with gene regulation such as, e.g., proteins associated
with chromatin (See, e.g., Dejardin and Kingston (2009) Cell
136:175), proteins that regulate (upregulate or downregulate)
methylation, proteins that regulate (upregulate or downregulate)
histone acetylation, proteins that regulate (upregulate or
downregulate) transcription, proteins that regulate (upregulate or
downregulate) post-transcriptional regulation, proteins that
regulate (upregulate or downregulate) RNA transport, proteins that
regulate (upregulate or downregulate) mRNA degradation, proteins
that regulate (upregulate or downregulate) translation, proteins
that regulate (upregulate or downregulate) post-translational
modifications and the like.
[0049] In certain aspects, a retrievable label is activatable. As
used herein, the term "activatable" refers to a retrievable label
that is inert (i.e., does not bind a target) until activated (e.g.,
by exposure of the activatable, retrievable label to light, heat,
one or more chemical compounds or the like). In other aspects, a
retrievable label can bind one or more targets without the need for
activation of the retrievable label.
[0050] In certain exemplary embodiments, a polynucleotide (e.g., an
Oligopaint) has a detectable label bound thereto. As used herein,
the term "detectable label" refers to a label that is attached to a
polynucleotide (e.g., an Oligopaint) and can be used to identify a
target (e.g., a chromosome or a sub-chromosomal region) to which
one or more Oligopaints have hybridized. Typically, a detectable
label is attached to the 3'- or 5'-end of a polynucleotide (e.g.,
an Oligopaint). Alternatively, a detectable label is attached to an
internal portion of an oligonucleotide (i.e., not at the 3' or the
5' end). Detectable labels may vary widely in size and
compositions; the following references provide guidance for
selecting oligonucleotide tags appropriate for particular
embodiments: Brenner, U.S. Pat. No. 5,635,400; Brenner et al.,
Proc. Natl. Acad. Sci., 97: 1665; Shoemaker et al. (1996) Nature
Genetics, 14:450; Morris et al., EP Patent Pub. 0799897A1; Wallace,
U.S. Pat. No. 5,981,179; and the like. In certain exemplary
embodiments, a polynucleotide (e.g., an Oligopaint) including one
or more detectable labels can have a length within a range of from
4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20
nucleotides, respectively. In other exemplary embodiments a
polynucleotide (e.g., an Oligopaint) including one or more
detectable labels can have a length of at least 30 nucleotides, at
least 40 nucleotides, at least 50 nucleotides, at least 60
nucleotides, at least 70 nucleotides, at least 80 nucleotides, at
least 90 nucleotides, at least 100 nucleotides, at least 150
nucleotides, at least 200 nucleotides, at least 300 nucleotides, at
least 400 nucleotides, at least 500 nucleotides, at least 600
nucleotides, at least 700 nucleotides, at least 800 nucleotides, at
least 900 nucleotides, at least 1000 nucleotides or greater.
[0051] Methods for incorporating detectable labels into nucleic
acid probes are well known. Typically, detectable labels (e.g., as
hapten- or fluorochrome-conjugated deoxyribonucleotides) are
incorporated into an oligopaint during a polymerization or
amplification step, e.g., by PCR, nick translation, random primer
labeling, terminal transferase tailing (e.g., one or more labels
can be added after cleavage of the primer sequence), and others
(see Ausubel et al., 1997, Current Protocols In Molecular Biology,
Greene Publishing and Wiley-Interscience, New York).
[0052] In certain aspects, a suitable retrievable label or
detectable label includes, but is not limited to, a capture moiety
such as a hydrophobic compound, an oligonucleotide, an antibody or
fragment of an antibody, a protein, a peptide, a chemical
cross-linker, an intercalator, a molecular cage (e.g., within a
cage or other structure, e.g., protein cages, fullerene cages,
zeolite cages, photon cages, and the like), or one or more elements
of a capture pair, e.g., biotin-avidin, biotin-streptavidin,
NHS-ester and the like, a thioether linkage, static charge
interactions, van der Waals forces and the like (See, e.g., Holtke
et al., U.S. Pat. Nos. 5,344,757; 5,702,888; and 5,354,657; Huber
et al., U.S. Pat. No. 5,198,537; Miyoshi, U.S. Pat. No. 4,849,336;
Misiura and Gait, PCT publication WO 91/17160). In certain aspects,
a suitable retrievable label or detectable label is an enzyme
(e.g., a methylase and/or a cleaving enzyme). In one aspect, an
antibody specific against the enzyme can be used to retrieve or
detect the enzyme and accordingly, retrieve or detect an
oligonucleotide sequence attached to the enzyme. In another aspect,
an antibody specific against the enzyme can be used to retrieve or
detect the enzyme and, after stringent washes, retrieve or detect
an first oligonucleotide sequence that is hybridized to a second
oligonucleotide sequence having the enzyme attached thereto.
[0053] Biotin, or a derivative thereof, may be used as an
oligonucleotide (e.g., Oligopaint) label (e.g., as a retrievable
label and/or a detectable label), and subsequently bound by a
avidin/streptavidin derivative (e.g., detectably labeled, e.g.,
phycoerythrin-conjugated streptavidin), or an anti-biotin antibody
(e.g., a detectably labeled antibody). Digoxigenin may be
incorporated as a label and subsequently bound by a detectably
labeled anti-digoxigenin antibody (e.g., a detectably labeled
antibody, e.g., fluoresceinated anti-digoxigenin). An
aminoallyl-dUTP residue may be incorporated into an oligonucleotide
and subsequently coupled to an N-hydroxy succinimide (NHS)
derivatized fluorescent dye, such as those listed infra. In
general, any member of a conjugate pair may be incorporated into a
retrievable label and/or a detectable label provided that a
detectably labeled conjugate partner can be bound to permit
detection. As used herein, the term antibody refers to an antibody
molecule of any class, or any sub-fragment thereof, such as an
Fab.
[0054] Other suitable labels (retrievable labels and/or detectable
labels) include, but are not limited to, fluorescein (FAM),
digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine
(BrdU), hexahistidine (6.times. His), phosphor-amino acids (e.g.
P-tyr, P-ser, P-thr) and the like. In one embodiment the following
hapten/antibody pairs are used for retrieval and/or detection:
biotin/.alpha.-biotin, digoxigenin/a-digoxigenin, dinitrophenol
(DNP)/.alpha.-DNP, 5-Carboxyfluorescein (FAM)/.alpha.-FAM.
[0055] Additional suitable labels (retrievable labels and/or
detectable labels) include, but are not limited to, chemical
cross-linking agents. Cross-linking agents typically contain at
least two reactive groups that are reactive towards numerous
groups, including, but not limited to, sulfhydryls and amines, and
create chemical covalent bonds between two or more molecules.
Functional groups that can be targeted with cross-linking agents
include, but are not limited to, primary amines, carboxyls,
sulfhydryls, carbohydrates and carboxylic acids. Protein molecules
have many of these functional groups and therefore proteins and
peptides can be readily conjugated using cross-linking agents.
Cross-linking agents are well known in the art and are commercially
available (Thermo Scientific (Rockford, Ill.)).
[0056] Fluorescent labels and their attachment to oligonucleotides
(e.g., to Oligopaints) are described in many reviews, including
Haugland, Handbook of Fluorescent Probes and Research Chemicals,
Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and
Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993);
Eckstein, editor, Oligonucleotides and Analogues: A Practical
Approach (IRL Press, Oxford, 1991); Wetmur, Critical Reviews in
Biochemistry and Molecular Biology, 26:227-259 (1991); and the
like. Particular methodologies applicable to the Oligopaint methods
and compositions described herein are disclosed in the following
sample of references: Fung et al., U.S. Pat. No. 4,757,141; Hobbs,
Jr., et al. U.S. Pat. No. 5,151,507; Cruickshank, U.S. Pat. No.
5,091,519. In one embodiment, one or more fluorescent dyes are used
as labels for Oligopaints, e.g., as disclosed by Menchen et al.,
U.S. Pat. No. 5,188,934 (4,7-dichlorofluorscein dyes); Begot et
al., U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine
dyes); Lee et al., U.S. Pat. No. 5,847,162 (4,7-dichlororhodamine
dyes); Khanna et al., U.S. Pat. No. 4,318,846 (ether-substituted
fluorescein dyes); Lee et al., U.S. Pat. No. 5,800,996 (energy
transfer dyes); Lee et al., U.S. Pat. No. 5,066,580 (xanthine
dyes): Mathies et al., U.S. Pat. No. 5,688,648 (energy transfer
dyes); and the like. Labelling can also be carried out with quantum
dots, as disclosed in the following patents and patent
publications: U.S. Pat. Nos. 6,322,901; 6,576,291; 6,423,551;
6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392;
2002/0045045; 2003/0017264; and the like. Amines can be
incorporated into Oligopaints, and labels can be added via the
amines using methods known in the art. As used herein, the term
"fluorescent label" includes a signaling moiety that conveys
information through the fluorescent absorption and/or emission
properties of one or more molecules. Such fluorescent properties
include fluorescence intensity, fluorescence life time, emission
spectrum characteristics, energy transfer and the like.
[0057] Commercially available fluorescent nucleotide analogues
readily incorporated into the Oligopaints include, for example,
Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences,
Piscataway, N.J.), fluorescein-12-dUTP,
tetramethylrhodamine-6-dUTP, TEXAS RED.TM.-5-dUTP, CASCADE
BLUE.TM.-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY
TMTR-14-dUTP, RHODAMINE GREEN.TM.-5-dUTP, OREGON GREENR.TM.
488-5-dUTP, TEXAS RED.TM.-12-dUTP, BODIPY TM 630/650-14-dUTP,
BODIPY TM 650/665-14-dUTP, ALEXA FLUOR.TM. 488-5-dUTP, ALEXA
FLUOR.TM. 532-5-dUTP, ALEXA FLUOR.TM. 568-5-dUTP, ALEXA FLUOR.TM.
594-5-dUTP, ALEXA FLUOR.TM. 546-14-dUTP, fluorescein-12-UTP,
tetramethylrhodamine-6-UTP, TEXAS RED.TM.-5-UTP, mCherry, CASCADE
BLUE.TM.-7-UTP, BODIPY TM FL-14-UTP, BODIPY TMR-14-UTP, BODIPY TM
TR-14-UTP, RHODAMINE GREEN.TM.-5-UTP, ALEXA FLUOR.TM. 488-5-UTP,
ALEXA FLUOR.TM. 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.).
Protocols are available for custom synthesis of nucleotides having
other fluorophores. Henegariu et al., "Custom
Fluorescent-Nucleotide Synthesis as an Alternative Method for
Nucleic Acid Labeling," Nature Biotechnol. 18:345-348 (2000).
[0058] Other fluorophores available for post-synthetic attachment
include, inter alia, ALEXA FLUOR.TM. 350, ALEXA FLUOR.TM. 532,
ALEXA FLUOR.TM. 546, ALEXA FLUOR.TM. 568, ALEXA FLUOR.TM. 594,
ALEXA FLUOR.TM. 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY
530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY
564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY
650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine
B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue,
rhodamine 6G, rhodamine green, rhodamine red, tetramethyl
rhodamine, DYLIGHT.TM. DYES (e.g., DYLIGHT.TM. 405, DYLIGHT.TM.
488, DYLIGHT.TM. 549, DYLIGHT.TM. 594, DYLIGHT.TM. 633, DYLIGHT.TM.
649, DYLIGHT.TM. 680, DYLIGHT.TM. 750, DYLIGHT.TM. 800 and the
like) (available from Thermo Fisher Scientific, Rockford, Ill.),
Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.),
and Cy2, Cy3.5, Cy5.5, and Cy7 (available from Amersham
Biosciences, Piscataway, N.J. USA, and others).
[0059] FRET tandem fluorophores may also be used, such as
PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7;
also, PE-Alexa dyes (610, 647, 680) and APC-Alexa dyes.
[0060] Metallic silver particles may be coated onto the surface of
the array to enhance signal from fluorescently labeled
oligonucleotide sequences bound to an array. Lakowicz et al. (2003)
BioTechniques 34:62.
[0061] Detection method(s) used will depend on the particular
detectable labels used in the Oligopaints. In certain exemplary
embodiments, chromosomes and/or chromosomal regions having one or
more Oligopaints bound thereto may be selected for and/or screened
for using a microscope, a spectrophotometer, a tube luminometer or
plate luminometer, x-ray film, a scintillator, a fluorescence
activated cell sorting (FACS) apparatus, a microfluidics apparatus
or the like.
[0062] When fluorescently labeled Oligopaints are used,
fluorescence photomicroscopy can be used to detect and record the
results of in situ hybridization using routine methods known in the
art. Alternatively, digital (computer implemented) fluorescence
microscopy with image-processing capability may be used. Two
well-known systems for imaging FISH of chromosomes having multiple
colored labels bound thereto include multiplex-FISH (M-FISH) and
spectral karyotyping (SKY). See Schrock et al. (1996) Science
273:494; Roberts et al. (1999) Genes Chrom. Cancer 25:241; Fransz
et al. (2002) Proc. Natl. Acad. Sci. USA 99:14584; Bayani et al.
(2004) Curr. Protocol. Cell Biol. 22.5.1-22.5.25; Danilova et al.
(2008) Chromosoma 117:345; U.S. Pat. No. 6,066,459; and FISH
TAG.TM. DNA Multicolor Kit instructions (Molecular probes) for a
review of methods for painting chromosomes and detecting painted
chromosomes.
[0063] In certain exemplary embodiments, images of fluorescently
labeled chromosomes are detected and recorded using a computerized
imaging system such as the Applied Imaging Corporation CytoVision
System (Applied Imaging Corporation, Santa Clara, Calif.) with
modifications (e.g., software, Chroma 84000 filter set, and an
enhanced filter wheel). Other suitable systems include a
computerized imaging system using a cooled CCD camera
(Photometrics, NU200 series equipped with Kodak KAF 1400 CCD)
coupled to a Zeiss Axiophot microscope, with images processed as
described by Ried et al. (1992) Proc. Natl. Acad. Sci. USA
89:1388). Other suitable imaging and analysis systems are described
by Schrock et al., supra; and Speicher et al., supra.
[0064] The in situ hybridization methods described herein can be
performed on a variety of biological or clinical samples, in cells
that are in any (or all) stage(s) of the cell cycle (e.g., mitosis,
meiosis, interphase, G0, G1, S and/or G2). Examples include all
types of cell culture, animal or plant tissue, peripheral blood
lymphocytes, buccal smears, touch preparations prepared from
uncultured primary tumors, cancer cells, bone marrow, cells
obtained from biopsy or cells in bodily fluids (e.g., blood, urine,
sputum and the like), cells from amniotic fluid, cells from
maternal blood (e.g., fetal cells), cells from testis and ovary,
and the like. Samples are prepared for assays of the invention
using conventional techniques, which typically depend on the source
from which a sample or specimen is taken. These examples are not to
be construed as limiting the sample types applicable to the methods
and/or compositions described herein.
[0065] In certain exemplary embodiments, Oligopaints include
multiple chromosome-specific probes, which are differentially
labeled (i.e., at least two of the chromosome-specific probes are
differently labeled). Various approaches to multi-color chromosome
painting have been described in the art and can be adapted to the
present invention following the guidance provided herein. Examples
of such differential labeling ("multicolor FISH") include those
described by Schrock et al. (1996) Science 273:494, and Speicher et
al. (1996) Nature Genet. 12:368). Schrock et al. describes a
spectral imaging method, in which epifluorescence filter sets and
computer software is used to detect and discriminate between
multiple differently labeled DNA probes hybridized simultaneously
to a target chromosome set. Speicher et al. describes using
different combinations of 5 fluorochromes to label each of the
human chromosomes (or chromosome arms) in a 27-color FISH termed
"combinatorial multifluor FISH"). Other suitable methods may also
be used (see, e.g., Ried et al., 1992, Proc. Natl. Acad. Sci. USA
89:1388-92).
[0066] Hybridization of the Oligopaints of the invention to target
chromosomes sequences can be accomplished by standard in situ
hybridization (ISH) techniques (see, e.g., Gall and Pardue (1981)
Meth. Enzymol. 21:470; Henderson (1982) Int. Review of Cytology
76:1). Generally, ISH comprises the following major steps: (1)
fixation of the biological structure to be analyzed (e.g., a
chromosome spread), (2) pre-hybridization treatment of the
biological structure to increase accessibility of target DNA (e.g.,
denaturation with heat or alkali), (3) optional pre-hybridization
treatment to reduce nonspecific binding (e.g., by blocking the
hybridization capacity of repetitive sequences), (4) hybridization
of the mixture of nucleic acids to the nucleic acid in the
biological structure or tissue; (5) post-hybridization washes to
remove nucleic acid fragments not bound in the hybridization and
(6) detection of the hybridized labelled oligonucleotides (e.g.,
hybridized Oligopaints). The reagents used in each of these steps
and their conditions of use vary depending on the particular
situation. For instance, step 3 will not always be necessary as the
Oligopaints described herein can be designed to avoid repetitive
sequences). Hybridization conditions are also described in U.S.
Pat. No. 5,447,841. It will be appreciated that numerous variations
of in situ hybridization protocols and conditions are known and may
be used in conjunction with the present invention by practitioners
following the guidance provided herein.
[0067] As used herein, the term "hybridization" refers to the
process in which two single-stranded polynucleotides bind
non-covalently to form a stable double-stranded polynucleotide. The
term "hybridization" may also refer to triple-stranded
hybridization. The resulting (usually) double-stranded
polynucleotide is a "hybrid" or "duplex." "Hybridization
conditions" will typically include salt concentrations of less than
about 1 M, more usually less than about 500 mM and even more
usually less than about 200 mM. Hybridization temperatures can be
as low as 5.degree. C., but are typically greater than 22.degree.
C., more typically greater than about 30.degree. C., and often in
excess of about 37.degree. C. Hybridizations are usually performed
under stringent conditions, i.e., conditions under which a probe
will hybridize to its target subsequence. Stringent conditions are
sequence-dependent and are different in different circumstances.
Longer fragments may require higher hybridization temperatures for
specific hybridization. As other factors may affect the stringency
of hybridization, including base composition and length of the
complementary strands, presence of organic solvents and extent of
base mismatching, the combination of parameters is more important
than the absolute measure of any one alone. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
T.sub.m for the specific sequence at s defined ionic strength and
pH. Exemplary stringent conditions include salt concentration of at
least 0.01 M to no more than 1 M Na ion concentration (or other
salts) at a pH 7.0 to 8.3 and a temperature of at least 25.degree.
C. For example, conditions of 5.times.SSPE (750 mM NaCl, 50 mM Na
phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30.degree. C.
are suitable for allele-specific probe hybridizations. For
stringent conditions, see for example, Sambrook, Fritsche and
Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed. Cold
Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization,
1.sup.st Ed., BIOS Scientific Publishers Limited (1999).
"Hybridizing specifically to" or "specifically hybridizing to" or
like expressions refer to the binding, duplexing, or hybridizing of
a molecule substantially to or only to a particular nucleotide
sequence or sequences under stringent conditions when that sequence
is present in a complex mixture (e.g., total cellular) DNA or
RNA.
[0068] In certain exemplary embodiments, synthesis of
oligonucleotides (e.g., Oligopaints) and/or amplification of
oligonucleotides (e.g., Oligopaints) can be performed using a
support. In certain aspects, multiple supports (tens, hundreds,
thousands or more) may be utilized (e.g., synthesized, amplified,
hybridized or the like) in parallel. Suitable supports include, but
are not limited to, slides (e.g., microscope slides), beads, chips,
particles, strands, gels, sheets, tubing (e.g., microfuge tubes,
test tubes, cuvettes), spheres, containers, capillaries,
microfibers, pads, slices, films, plates (e.g., multi-well plates),
microfluidic supports (e.g., microarray chips, flow channel plates,
biochips and the like) and the like. In various embodiments, the
solid supports may be biological, nonbiological, organic, inorganic
or combinations thereof. When using supports that are substantially
planar, the support may be physically separated into regions, for
example, with trenches, grooves, wells, or chemical barriers (e.g.,
lacking a lipid-binding coating). In exemplary embodiments,
supports can be made of a variety of materials including, but not
limited to glass, quartz, ceramic, plastic, polystyrene,
methylstyrene, acrylic polymers, titanium, latex, sepharose,
cellulose, nylon and the like and any combination thereof. Such
supports and their uses are well known in the art.
[0069] In certain exemplary embodiments, supports may have
functional groups on their surface which can be used to attach a
lipid bilayer (e.g., a phospholipid bilayer) to the support. For
example, at least a portion of the support can be coated with
silane and dextran (e.g., high molecular weight dextran). Dextran
in its hydrated form can function as a molecular cushion for the
membrane and is capable of binding lipids on the support. Suitable
functional groups include, but are not limited to, silicon oxides
(e.g., SiO.sub.2), MgF.sub.2, CaF.sub.2, mica, polyacrylamide,
dextran and the like and any combination thereof.
[0070] In certain exemplary embodiments, methods of generating and
amplifying synthetic oligonucleotide sequences, e.g., Oligopaint
sequences, are provided. As used herein, the term "oligonucleotide"
is intended to include, but is not limited to, a single-stranded
DNA or RNA molecule, typically prepared by synthetic means.
Nucleotides of the present invention will typically be the
naturally-occurring nucleotides such as nucleotides derived from
adenosine, guanosine, uridine, cytidine and thymidine. When
oligonucleotides are referred to as "double-stranded," it is
understood by those of skill in the art that a pair of
oligonucleotides exists in a hydrogen-bonded, helical array
typically associated with, for example, DNA. In addition to the
100% complementary form of double-stranded oligonucleotides, the
term "double-stranded" as used herein is also meant to include
those form which include such structural features as bulges and
loops (see Stryer, Biochemistry, Third Ed. (1988), incorporated
herein by reference in its entirety for all purposes). As used
herein, the term "polynucleotide" is intended to include, but is
not limited to, two or more oligonucleotides joined together (e.g.,
by hybridization, ligation, polymerization and the like).
[0071] The term "operably linked," when describing the relationship
between two nucleic acid regions, refers to a juxtaposition wherein
the regions are in a relationship permitting them to function in
their intended manner. For example, a control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences, such as when the appropriate
molecules (e.g., inducers and polymerases) are bound to the control
or regulatory sequence(s).
[0072] In certain exemplary embodiments, nucleotide analogs or
derivatives will be used, such as nucleosides or nucleotides having
protecting groups on either the base portion or sugar portion of
the molecule, or having attached or incorporated labels, or
isosteric replacements which result in monomers that behave in
either a synthetic or physiological environment in a manner similar
to the parent monomer. The nucleotides can have a protecting group
which is linked to, and masks, a reactive group on the nucleotide.
A variety of protecting groups are useful in the invention and can
be selected depending on the synthesis techniques employed and are
discussed further below. After the nucleotide is attached to the
support or growing nucleic acid, the protecting group can be
removed.
[0073] Oligonucleotides or fragments thereof may be purchased from
commercial sources. Oligonucleotide sequences may be prepared by
any suitable method, e.g., the phosphoramidite method described by
Beaucage and Carruthers ((1981) Tetrahedron Lett. 22: 1859) or the
triester method according to Matteucci et al. (1981) J. Am. Chem.
Soc. 103:3185), both incorporated herein by reference in their
entirety for all purposes, or by other chemical methods using
either a commercial automated oligonucleotide synthesizer or
high-throughput, high-density array methods described herein and
known in the art (see U.S. Pat. Nos. 5,602,244, 5,574,146,
5,554,744, 5,428,148, 5,264,566, 5,141,813, 5,959,463, 4,861,571
and 4,659,774, incorporated herein by reference in its entirety for
all purposes). Pre-synthesized oligonucleotides and chips
containing oligonucleotides may also be obtained commercially from
a variety of vendors.
[0074] In an exemplary embodiment, construction and/or selection
oligonucleotides may be synthesized on a solid support using
maskless array synthesizer (MAS). Maskless array synthesizers are
described, for example, in PCT application No. WO 99/42813 and in
corresponding U.S. Pat. No. 6,375,903. Other examples are known of
maskless instruments which can fabricate a custom DNA microarray in
which each of the features in the array has a single stranded DNA
molecule of desired sequence. An exemplary type of instrument is
the type shown in FIG. 5 of U.S. Pat. No. 6,375,903, based on the
use of reflective optics. It is a desirable that this type of
maskless array synthesizer is under software control. Since the
entire process of microarray synthesis can be accomplished in only
a few hours, and since suitable software permits the desired DNA
sequences to be altered at will, this class of device makes it
possible to fabricate microarrays including DNA segments of
different sequence every day or even multiple times per day on one
instrument. The differences in DNA sequence of the DNA segments in
the microarray can also be slight or dramatic, it makes no
difference to the process. The MAS instrument may be used in the
form it would normally be used to make microarrays for
hybridization experiments, but it may also be adapted to have
features specifically adapted for the compositions, methods, and
systems described herein. For example, it may be desirable to
substitute a coherent light source, i.e., a laser, for the light
source shown in FIG. 5 of the above-mentioned U.S. Pat. No.
6,375,903. If a laser is used as the light source, a beam expanded
and scatter plate may be used after the laser to transform the
narrow light beam from the laser into a broader light source to
illuminate the micromirror arrays used in the maskless array
synthesizer. It is also envisioned that changes may be made to the
flow cell in which the microarray is synthesized. In particular, it
is envisioned that the flow cell can be compartmentalized, with
linear rows of array elements being in fluid communication with
each other by a common fluid channel, but each channel being
separated from adjacent channels associated with neighboring rows
of array elements. During microarray synthesis, the channels all
receive the same fluids at the same time. After the DNA segments
are separated from the substrate, the channels serve to permit the
DNA segments from the row of array elements to congregate with each
other and begin to self-assemble by hybridization.
[0075] Other methods for synthesizing oligonucleotides (e.g.,
Oligopaints) include, for example, light-directed methods utilizing
masks, flow channel methods, spotting methods, pin-based methods,
and methods utilizing multiple supports.
[0076] Light directed methods utilizing masks (e.g., VLSIPS.TM.
methods) for the synthesis of oligonucleotides is described, for
example, in U.S. Pat. Nos. 5,143,854, 5,510,270 and 5,527,681.
These methods involve activating predefined regions of a solid
support and then contacting the support with a preselected monomer
solution. Selected regions can be activated by irradiation with a
light source through a mask much in the manner of photolithography
techniques used in integrated circuit fabrication. Other regions of
the support remain inactive because illumination is blocked by the
mask and they remain chemically protected. Thus, a light pattern
defines which regions of the support react with a given monomer. By
repeatedly activating different sets of predefined regions and
contacting different monomer solutions with the support, a diverse
array of polymers is produced on the support. Other steps, such as
washing unreacted monomer solution from the support, can be used as
necessary. Other applicable methods include mechanical techniques
such as those described in U.S. Pat. No. 5,384,261.
[0077] Additional methods applicable to synthesis and/or
amplification of oligonucleotides (e.g., Oligopaints) on a single
support are described, for example, in U.S. Pat. No. 5,384,261. For
example reagents may be delivered to the support by either (1)
flowing within a channel defined on predefined regions or (2)
"spotting" on predefined regions. Other approaches, as well as
combinations of spotting and flowing, may be employed as well. In
each instance, certain activated regions of the support are
mechanically separated from other regions when the monomer
solutions are delivered to the various reaction sites.
[0078] Flow channel methods involve, for example, microfluidic
systems to control synthesis of oligonucleotides on a solid
support. For example, diverse polymer sequences may be synthesized
at selected regions of a solid support by forming flow channels on
a surface of the support through which appropriate reagents flow or
in which appropriate reagents are placed. One of skill in the art
will recognize that there are alternative methods of forming
channels or otherwise protecting a portion of the surface of the
support. For example, a protective coating such as a hydrophilic or
hydrophobic coating (depending upon the nature of the solvent) is
utilized over portions of the support to be protected, sometimes in
combination with materials that facilitate wetting by the reactant
solution in other regions. In this manner, the flowing solutions
are further prevented from passing outside of their designated flow
paths.
[0079] Spotting methods for preparation of oligonucleotides on a
solid support involve delivering reactants in relatively small
quantities by directly depositing them in selected regions. In some
steps, the entire support surface can be sprayed or otherwise
coated with a solution, if it is more efficient to do so. Precisely
measured aliquots of monomer solutions may be deposited dropwise by
a dispenser that moves from region to region. Typical dispensers
include a micropipette to deliver the monomer solution to the
support and a robotic system to control the position of the
micropipette with respect to the support, or an ink jet printer. In
other embodiments, the dispenser includes a series of tubes, a
manifold, an array of pipettes, or the like so that various
reagents can be delivered to the reaction regions
simultaneously.
[0080] Pin-based methods for synthesis of oligonucleotides on a
solid support are described, for example, in U.S. Pat. No.
5,288,514. Pin-based methods utilize a support having a plurality
of pins or other extensions. The pins are each inserted
simultaneously into individual reagent containers in a tray. An
array of 96 pins is commonly utilized with a 96-container tray,
such as a 96-well microtitre dish. Each tray is filled with a
particular reagent for coupling in a particular chemical reaction
on an individual pin. Accordingly, the trays will often contain
different reagents. Since the chemical reactions have been
optimized such that each of the reactions can be performed under a
relatively similar set of reaction conditions, it becomes possible
to conduct multiple chemical coupling steps simultaneously.
[0081] In yet another embodiment, a plurality of oligonucleotides
(e.g., Oligopaints) may be synthesized on multiple supports. One
example is a bead based synthesis method which is described, for
example, in U.S. Pat. Nos. 5,770,358, 5,639,603, and 5,541,061. For
the synthesis of molecules such as oligonucleotides on beads, a
large plurality of beads are suspended in a suitable carrier (such
as water) in a container. The beads are provided with optional
spacer molecules having an active site to which is complexed,
optionally, a protecting group. At each step of the synthesis, the
beads are divided for coupling into a plurality of containers.
After the nascent oligonucleotide chains are deprotected, a
different monomer solution is added to each container, so that on
all beads in a given container, the same nucleotide addition
reaction occurs. The beads are then washed of excess reagents,
pooled in a single container, mixed and re-distributed into another
plurality of containers in preparation for the next round of
synthesis. It should be noted that by virtue of the large number of
beads utilized at the outset, there will similarly be a large
number of beads randomly dispersed in the container, each having a
unique oligonucleotide sequence synthesized on a surface thereof
after numerous rounds of randomized addition of bases. An
individual bead may be tagged with a sequence which is unique to
the double-stranded oligonucleotide thereon, to allow for
identification during use.
[0082] In certain embodiments, a plurality of oligonucleotides
(e.g., Oligopaints) may be synthesized, amplified and/or used in
conjunction with beads and/or bead-based arrays. As used herein,
the term "bead" refers to a discrete particle that may be spherical
(e.g., microspheres) or have an irregular shape. Beads may be as
small as approximately 0.1 .mu.m in diameter or as large
approximately several millimeters in diameter. Beads typically
range in size from approximately 0.1 .mu.m to 200 .mu.m in
diameter. Beads may comprise a variety of materials including, but
not limited to, paramagnetic materials, ceramic, plastic, glass,
polystyrene, methylstyrene, acrylic polymers, titanium, latex,
sepharose, cellulose, nylon and the like.
[0083] In certain aspects, beads may have functional groups on
their surface which can be used to oligonucleotides (e.g.,
Oligopaints) to the bead. Oligonucleotide sequences can be attached
to a bead by hybridization (e.g., binding to a polymer), covalent
attachment, magnetic attachment, affinity attachment and the like.
For example, the bead can be coated with streptavidin and the
nucleic acid sequence can include a biotin moiety. The biotin is
capable of binding streptavidin on the bead, thus attaching the
nucleic acid sequence to the bead. Beads coated with streptavidin,
oligo-dT, and histidine tag binding substrate are commercially
available (Dynal Biotech, Brown Deer, Wis.). Beads may also be
functionalized using, for example, solid-phase chemistries known in
the art, such as those for generating nucleic acid arrays, such as
carboxyl, amino, and hydroxyl groups, or functionalized silicon
compounds (see, for example, U.S. Pat. No. 5,919,523).
[0084] Various exemplary protecting groups useful for synthesis of
oligonucleotides on a solid support are described in, for example,
Atherton et al., 1989, Solid Phase Peptide Synthesis, IRL Press. In
various embodiments, the methods described herein utilize solid
supports for immobilization of nucleic acids. For example,
oligonucleotides may be synthesized on one or more solid supports.
Exemplary solid supports include, for example, slides, beads,
chips, particles, strands, gels, sheets, tubing, spheres,
containers, capillaries, pads, slices, films, or plates. In various
embodiments, the solid supports may be biological, nonbiological,
organic, inorganic, or combinations thereof. When using supports
that are substantially planar, the support may be physically
separated into regions, for example, with trenches, grooves, wells,
or chemical barriers (e.g., hydrophobic coatings, etc.). Supports
that are transparent to light are useful when the assay involves
optical detection (see e.g., U.S. Pat. No. 5,545,531). The surface
of the solid support will typically contain reactive groups, such
as carboxyl, amino, and hydroxyl or may be coated with
functionalized silicon compounds (see e.g., U.S. Pat. No.
5,919,523).
[0085] In one embodiment, the oligonucleotides synthesized on the
solid support may be used as a template for the production of
Oligopaints. For example, the support bound oligonucleotides may be
contacted with primers that hybridize to the oligonucleotides under
conditions that permit chain extension of the primers. The support
bound duplexes may then be denatured, pooled and subjected to
further rounds of amplification to produce Oligopaints in solution.
In another embodiment, the support-bound oligonucleotides may be
removed from the solid, pooled and amplified to produce Oligopaints
in solution. The oligonucleotides may be removed from the solid
support, for example, by exposure to conditions such as acid, base,
oxidation, reduction, heat, light, metal ion catalysis,
displacement or elimination chemistry, or by enzymatic
cleavage.
[0086] In one embodiment, oligonucleotides may be attached to a
solid support through a cleavable linkage moiety. For example, the
solid support may be functionalized to provide cleavable linkers
for covalent attachment to the oligonucleotides. The linker moiety
may be one, two, three, four, five, six or more atoms in length.
Alternatively, the cleavable moiety may be within an
oligonucleotide and may be introduced during in situ synthesis. A
broad variety of cleavable moieties are available in the art of
solid phase and microarray oligonucleotide synthesis (see e.g.,
Pon, R., Methods Mol. Biol. 20:465-496 (1993); Verma et al., Ann.
Rev. Biochem. 67:99-134 (1998); U.S. Pat. Nos. 5,739,386, 5,700,642
and 5,830,655; and U.S. Patent Publication Nos. 2003/0186226 and
2004/0106728). A suitable cleavable moiety may be selected to be
compatible with the nature of the protecting group of the
nucleoside bases, the choice of solid support, and/or the mode of
reagent delivery, among others. In an exemplary embodiment, the
oligonucleotides cleaved from the solid support contain a free
3'-OH end. Alternatively, the free 3'-OH end may also be obtained
by chemical or enzymatic treatment, following the cleavage of
oligonucleotides. The cleavable moiety may be removed under
conditions which do not degrade the oligonucleotides. The linker
may be cleaved using two approaches, either (a) simultaneously
under the same conditions as the deprotection step or (b)
subsequently utilizing a different condition or reagent for linker
cleavage after the completion of the deprotection step.
[0087] The covalent immobilization site may either be at the 5' end
of the oligonucleotide or at the 3' end of the oligonucleotide. In
some instances, the immobilization site may be within the
oligonucleotide (i.e. at a site other than the 5' or 3' end of the
oligonucleotide). The cleavable site may be located along the
oligonucleotide backbone, for example, a modified 3'-5'
internucleotide linkage in place of one of the phosphodiester
groups, such as ribose, dialkoxysilane, phosphorothioate, and
phosphoramidate internucleotide linkage. The cleavable
oligonucleotide analogs may also include a substituent on, or
replacement of, one of the bases or sugars, such as
7-deazaguanosine, 5-methylcytosine, inosine, uridine, and the
like.
[0088] In one embodiment, cleavable sites contained within the
modified oligonucleotide may include chemically cleavable groups,
such as dialkoxysilane, 3'-(S)-phosphorothioate,
5'-(S)-phosphorothioate, 3'-(N)-phosphoramidate,
5'-(N)phosphoramidate, and ribose. Synthesis and cleavage
conditions of chemically cleavable oligonucleotides are described
in U.S. Pat. Nos. 5,700,642 and 5,830,655. For example, depending
upon the choice of cleavable site to be introduced, either a
functionalized nucleoside or a modified nucleoside dimer may be
first prepared, and then selectively introduced into a growing
oligonucleotide fragment during the course of oligonucleotide
synthesis. Selective cleavage of the dialkoxysilane may be effected
by treatment with fluoride ion. Phosphorothioate internucleotide
linkage may be selectively cleaved under mild oxidative conditions.
Selective cleavage of the phosphoramidate bond may be carried out
under mild acid conditions, such as 80% acetic acid. Selective
cleavage of ribose may be carried out by treatment with dilute
ammonium hydroxide.
[0089] In another embodiment, a non-cleavable hydroxyl linker may
be converted into a cleavable linker by coupling a special
phosphoramidite to the hydroxyl group prior to the phosphoramidite
or H-phosphonate oligonucleotide synthesis as described in U.S.
Patent Application Publication No. 2003/0186226. The cleavage of
the chemical phosphorylation agent at the completion of the
oligonucleotide synthesis yields an oligonucleotide bearing a
phosphate group at the 3' end. The 3'-phosphate end may be
converted to a 3' hydroxyl end by a treatment with a chemical or an
enzyme, such as alkaline phosphatase, which is routinely carried
out by those skilled in the art.
[0090] In another embodiment, the cleavable linking moiety may be a
TOPS (two oligonucleotides per synthesis) linker (see e.g., PCT
publication WO 93/20092). For example, the TOPS phosphoramidite may
be used to convert a non-cleavable hydroxyl group on the solid
support to a cleavable linker. A preferred embodiment of TOPS
reagents is the Universal TOPS.TM. phosphoramidite. Conditions for
Universal TOPS.TM. phosphoramidite preparation, coupling and
cleavage are detailed, for example, in Hardy et al, Nucleic Acids
Research 22(15):2998-3004 (1994). The Universal TOPS.TM.
phosphoramidite yields a cyclic 3' phosphate that may be removed
under basic conditions, such as the extended ammonia and/or
ammonia/methylamine treatment, resulting in the natural 3' hydroxy
oligonucleotide.
[0091] In another embodiment, a cleavable linking moiety may be an
amino linker. The resulting oligonucleotides bound to the linker
via a phosphoramidite linkage may be cleaved with 80% acetic acid
yielding a 3'-phosphorylated oligonucleotide.
[0092] In another embodiment, the cleavable linking moiety may be a
photocleavable linker, such as an ortho-nitrobenzyl photocleavable
linker. Synthesis and cleavage conditions of photolabile
oligonucleotides on solid supports are described, for example, in
Venkatesan et al. J. of Org. Chem. 61:525-529 (1996), Kahl et al.,
J. of Org. Chem. 64:507-510 (1999), Kahl et al., J. of Org. Chem.
63:4870-4871 (1998), Greenberg et al., J. of Org. Chem. 59:746-753
(1994), Holmes et al., J. of Org. Chem. 62:2370-2380 (1997), and
U.S. Pat. No. 5,739,386. Ortho-nitobenzyl-based linkers, such as
hydroxymethyl, hydroxyethyl, and Fmoc-aminoethyl carboxylic acid
linkers, may also be obtained commercially.
[0093] In another embodiment, oligonucleotides may be removed from
a solid support by an enzyme such as a nuclease. For example,
oligonucleotides may be removed from a solid support upon exposure
to one or more restriction endonucleases, including, for example,
class IIs restriction enzymes. A restriction endonuclease
recognition sequence may be incorporated into the immobilized
oligonucleotides and the oligonucleotides may be contacted with one
or more restriction endonucleases to remove the oligonucleotides
from the support. In various embodiments, when using enzymatic
cleavage to remove the oligonucleotides from the support, it may be
desirable to contact the single stranded immobilized
oligonucleotides with primers, polymerase and dNTPs to form
immobilized duplexes. The duplexes may then be contacted with the
enzyme (e.g., a restriction endonuclease) to remove the duplexes
from the surface of the support. Methods for synthesizing a second
strand on a support bound oligonucleotide and methods for enzymatic
removal of support bound duplexes are described, for example, in
U.S. Pat. No. 6,326,489. Alternatively, short oligonucleotides that
are complementary to the restriction endonuclease recognition
and/or cleavage site (e.g., but are not complementary to the entire
support bound oligonucleotide) may be added to the support bound
oligonucleotides under hybridization conditions to facilitate
cleavage by a restriction endonuclease (see e.g., PCT Publication
No. WO 04/024886).
[0094] In yet another embodiment, a plurality of oligonucleotides
(e.g., Oligopaints) may be synthesized and/or amplified in
solution. Methods of synthesizing oligonucleotide sequences are
well-known in the art (See, e.g., Seliger (1993) Protocols for
Oligonucleotides and Analogs: Synthesis and Properties, vol. 20,
pp. 391-435, Efimov (2007) Nucleosides, Nucleotides & Nucleic
Acids 26:8, McMinn et al. (1997) J. Org. Chem. 62:7074, Froehler et
al. (1986) Nucleic Acids Res. 14:5399, Garegg (1986) Tet. Lett.
27:4051, Efimov (1983) Nucleic Acids Res. 11:8369, Reese (1978)
Tetrahedron 34:3143).
[0095] In certain embodiments, oligonucleotides (e.g., Oligopaints)
are double stranded (ds). In certain aspects, a ds oligonucleotide
may be synthesized as two single stranded oligonucleotides that are
hybridized together, thus forming a ds oligonucleotide.
Alternatively, a ds oligonucleotide may be synthesized is a ds form
(e.g., using a ss oligonucleotide as a template). In other
embodiments, oligonucleotides (e.g., Oligopaints) are single
stranded (ss). In certain aspects, a ss oligonucleotide is
generated in a ss form. In other aspects, a ss oligonucleotide is
synthesized in a ds form and is converted to ss form subsequent to
synthesis using any of a variety of methods well known in the art
(e.g., by incorporating dUs into the ds oligonucleotide during
synthesis that can be cleaved after synthesis, by chemical cleavage
after synthesis, by enzymatic cleavage after synthesis, by nuclease
digestion after synthesis, by light based cleavage after synthesis
and the like).
[0096] Exemplary chemically cleavable internucleotide linkages for
use in the methods described herein include, for example,
.beta.-cyano ether, 5'-deoxy-5'-aminocarbamate,
3'deoxy-3'-aminocarbamate, urea, 2'cyano-3',5'-phosphodiester,
3'-(S)-phosphorothioate, 5'-(S)-phosphorothioate,
3'-(N)-phosphoramidate, 5'-(N)-phosphoramidate, .alpha.-amino
amide, vicinal diol, ribonucleoside insertion,
2'-amino-3',5'-phosphodiester, allylic sulfoxide, ester, silyl
ether, dithioacetal, 5'-thio-furmal,
.alpha.-hydroxy-methyl-phosphonic bisamide, acetal, 3'-thio-furmal,
methylphosphonate and phosphotriester. Internucleoside silyl groups
such as trialkylsilyl ether and dialkoxysilane are cleaved by
treatment with fluoride ion. Base-cleavable sites include
.beta.-cyano ether, 5'-deoxy-5'-aminocarbamate,
3'-deoxy-3'-aminocarbamate, urea, 2'-cyano-3',5'-phosphodiester,
2'-amino-3',5'-phosphodiester, ester and ribose. Thio-containing
internucleotide bonds such as 3'-(S)-phosphorothioate and
5'-(S)-phosphorothioate are cleaved by treatment with silver
nitrate or mercuric chloride. Acid cleavable sites include
3'-(N)-phosphoramidate, 5'-(N)-phosphoramidate, dithioacetal,
acetal and phosphonic bisamide. An .alpha.-aminoamide
internucleoside bond is cleavable by treatment with isothiocyanate,
and titanium may be used to cleave a
2'-amino-3',5'-phosphodiester-O-ortho-benzyl internucleoside bond.
Vicinal diol linkages are cleavable by treatment with periodate.
Thermally cleavable groups include allylic sulfoxide and
cyclohexene while photo-labile linkages include nitrobenzylether
and thymidine dimer. Methods synthesizing and cleaving nucleic
acids containing chemically cleavable, thermally cleavable, and
photo-labile groups are described for example, in U.S. Pat. No.
5,700,642.
[0097] Enzymatic cleavage may be mediated by including a
restriction endonuclease cleavage site in the oligonucleotide
sequence. After synthesis of a ds oligonucleotide, the ds
oligonucleotide may be contacted with one or more endonucleases to
remove one strand. A wide variety of restriction endonucleases
having specific binding and/or cleavage sites are commercially
available, for example, from New England Biolabs (Ipswich,
Mass.).
[0098] In various embodiments, the methods disclosed herein
comprise amplification of oligonucleotide sequences including, for
example, Oligopaints. Amplification methods may comprise contacting
a nucleic acid with one or more primers that specifically hybridize
to the nucleic acid under conditions that facilitate hybridization
and chain extension. Exemplary methods for amplifying nucleic acids
include the polymerase chain reaction (PCR) (see, e.g., Mullis et
al. (1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263 and
Cleary et al. (2004) Nature Methods 1:241; and U.S. Pat. Nos.
4,683,195 and 4,683,202), anchor PCR, RACE PCR, ligation chain
reaction (LCR) (see, e.g., Landegran et al. (1988) Science
241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci.
U.S.A. 91:360-364), self sustained sequence replication (Guatelli
et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:1874),
transcriptional amplification system (Kwoh et al. (1989) Proc.
Natl. Acad. Sci. U.S.A. 86:1173), Q-Beta Replicase (Lizardi et al.
(1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000) J.
Biol. Chem. 275:2619; and Williams et al. (2002) J. Biol. Chem.
277:7790), the amplification methods described in U.S. Pat. Nos.
6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and
5,612,199, or any other nucleic acid amplification method using
techniques well known to those of skill in the art. In exemplary
embodiments, the methods disclosed herein utilize PCR
amplification.
[0099] In certain exemplary embodiments, universal primers will be
used to amplify nucleic acid sequences such as, for example,
Oligopaints. The term "universal primers" refers to a set of
primers (e.g., a forward and reverse primer) that may be used for
chain extension/amplification of a plurality of polynucleotides,
e.g., the primers hybridize to sites that are common to a plurality
of polynucleotides. For example, universal primers may be used for
amplification of all, or essentially all, polynucleotides in a
single pool. In certain aspects, forward primers and reverse
primers have the same sequence. In other aspects, the sequence of
forward primers differs from the sequence of reverse primers. In
still other aspects, a plurality of universal primers are provided,
e.g., tens, hundreds, thousands or more.
[0100] In certain embodiments, the universal primers may be
temporary primers that may be removed after amplification via
enzymatic or chemical cleavage. In certain embodiments, the
universal primers may be temporary primers that may be removed
after amplification via enzymatic or chemical cleavage. In other
embodiments, the universal primers may comprise a modification that
becomes incorporated into the polynucleotide molecules upon chain
extension. Exemplary modifications include, for example, a 3' or 5'
end cap, a label (e.g., fluorescein), or a tag (e.g., a tag that
facilitates immobilization or isolation of the polynucleotide, such
as, biotin, etc.).
[0101] In exemplary embodiments, primers may be designed to be
temporary to permit removal of the primers. Temporary primers may
be designed so as to be removable by chemical, thermal, light
based, or enzymatic cleavage. Cleavage may occur upon addition of
an external factor (e.g., an enzyme, chemical, heat, light, etc.)
or may occur automatically after a certain time period (e.g., after
n rounds of amplification). In one embodiment, temporary primers
may be removed by chemical cleavage. For example, primers having
acid labile or base labile sites may be used for amplification. The
amplified pool may then be exposed to acid or base to remove the
primer at the desired location. Alternatively, the temporary
primers may be removed by exposure to heat and/or light. For
example, primers having heat labile or photolabile sites may be
used for amplification. The amplified pool may then be exposed to
heat and/or light to remove the primer/primer binding sites at the
desired location. In another embodiment, an RNA primer may be used
for amplification thereby forming short stretches of RNA/DNA
hybrids at the ends of the nucleic acid molecule. The primer site
may then be removed by exposure to an RNase (e.g., RNase H). In
various embodiments, the method for removing the primer may only
cleave a single strand of the amplified duplex thereby leaving 3'
or 5' overhangs. Such overhangs may be removed using an exonuclease
to form blunt ended double stranded duplexes. For example,
RecJ.sub.f may be used to remove single stranded 5' overhangs and
Exonuclease I or Exonuclease T may be used to remove single
stranded 3' overhangs. Additionally, S.sub.1 nuclease, P.sub.1
nuclease, mung bean nuclease, and CEL I nuclease, may be used to
remove single stranded regions from a nucleic acid molecule.
RecJ.sub.f, Exonuclease I, Exonuclease T, and mung bean nuclease
are commercially available, for example, from New England Biolabs
(Ipswich, Mass.). S1 nuclease, P1 nuclease and CEL I nuclease are
described, for example, in Vogt, V. M., Eur. J. Biochem., 33:
192-200 (1973); Fujimoto et al., Agric. Biol. Chem. 38: 777-783
(1974); Vogt, V. M., Methods Enzymol. 65: 248-255 (1980); and Yang
et al., Biochemistry 39: 3533-3541 (2000).
[0102] In one embodiment, the temporary primers may be removed from
a nucleic acid by chemical, thermal, or light based cleavage as
described supra. In other embodiments, primers may be removed using
enzymatic cleavage. For example, primers may be designed to include
a restriction endonuclease cleavage site. After amplification, the
pool of nucleic acids may be contacted with one or more
endonucleases to produce double stranded breaks thereby removing
the primers. In certain embodiments, the forward and reverse
primers may be removed by the same or different restriction
endonucleases. Any type of restriction endonuclease may be used to
remove the primers/primer binding sites from nucleic acid
sequences. In various embodiments, restriction endonucleases that
produce 3' overhangs, 5' overhangs or blunt ends may be used.
[0103] In certain embodiments, it may be desirable to utilize a
primer comprising one or more modifications such as a cap (e.g., to
prevent exonuclease cleavage), a linking moiety (such as those
described above to facilitate immobilization of an oligonucleotide
onto a substrate), or a label (e.g., to facilitate detection,
isolation and/or immobilization of a nucleic acid construct).
Suitable modifications include, for example, various enzymes,
prosthetic groups, luminescent markers, bioluminescent markers,
fluorescent markers (e.g., fluorescein), radiolabels (e.g.,
.sup.32P, .sup.35S, etc.), biotin, polypeptide epitopes, etc. as
described further herein.
[0104] Embodiments of the present invention are directed to
oligonucleotide sequences (e.g., Oligopaints) having one or more
amplification sequences or amplification sites. As used herein, the
term "amplification site" is intended to include, but is not
limited to, a nucleic acid sequence located at the 5' and/or 3' end
of the oligonucleotide sequences of the present invention which
hybridizes a complementary nucleic acid sequence. In one aspect of
the invention, an amplification site is removed from the
oligonucleotide after amplification. In another aspect of the
invention, an amplification site includes one or more restriction
endonuclease recognition sequences recognized by one or more
restriction enzymes. In another aspect, an amplification site is
heat labile and/or photo labile and is cleavable by heat or light,
respectively. In yet another aspect, an amplification site is a
ribonucleic acid sequence cleavable by RNase. In still another
aspect, an amplification site is chemically cleavable (e.g., using
acid and/or base).
[0105] As used herein, the term "restriction endonuclease
recognition site" is intended to include, but is not limited to, a
particular nucleic acid sequence to which one or more restriction
enzymes bind, resulting in cleavage of a DNA molecule either at the
restriction endonuclease recognition sequence itself, or at a
sequence distal to the restriction endonuclease recognition
sequence. Restriction enzymes include, but are not limited to, type
I enzymes, type II enzymes, type IIS enzymes, type III enzymes and
type IV enzymes. The REBASE database provides a comprehensive
database of information about restriction enzymes, DNA
methyltransferases and related proteins involved in
restriction-modification. It contains both published and
unpublished work with information about restriction endonuclease
recognition sites and restriction endonuclease cleavage sites,
isoschizomers, commercial availability, crystal and sequence data
(see Roberts et al. (2005) Nucl. Acids Res. 33:D230, incorporated
herein by reference in its entirety for all purposes).
[0106] In certain aspects, primers of the present invention include
one or more restriction endonuclease recognition sites that enable
type IIS enzymes to cleave the nucleic acid several base pairs 3'
to the restriction endonuclease recognition sequence. As used
herein, the term "type IIS" refers to a restriction enzyme that
cuts at a site remote from its recognition sequence. Type IIS
enzymes are known to cut at a distances from their recognition
sites ranging from 0 to 20 base pairs. Examples of Type IIs
endonucleases include, for example, enzymes that produce a 3'
overhang, such as, for example, Bsr I, Bsm I, BstF5 I, BsrD I, Bts
I, Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX
I, Bcg I, Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 I, Eco57M
I, Gsu I, Ppi I, and Psr I; enzymes that produce a 5' overhang such
as, for example, BsmA I, Ple I, Fau I, Sap I, BspM I, SfaN I, Hga
I, Bvb I, Fok I, BceA I, BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I;
and enzymes that produce a blunt end, such as, for example, Mly I
and Btr I. Type-IIs endonucleases are commercially available and
are well known in the art (New England Biolabs, Ipswich, Mass.).
Information about the recognition sites, cut sites and conditions
for digestion using type IIs endonucleases may be found, for
example, on the Worldwide Web at
neb.com/nebecomm/enzymefindersearch bytypeIIs.asp). Restriction
endonuclease sequences and restriction enzymes are well known in
the art and restriction enzymes are commercially available (New
England Biolabs).
[0107] Certain exemplary embodiments are directed to the use of
computer software to automate design and/or interpretation of
genomic spacings, repeat-discriminating SNPs and/or colors for each
specific oligopaint set. Such software may be used in conjunction
with individuals performing interpretation by hand or in a
semi-automated fashion or combined with an automated system. In at
least some embodiments, the design and/or interpretation software
is implemented in a program written in the JAVA programming
language. The program may be compiled into an executable that may
then be run from a command prompt in the WINDOWS XP operating
system. Unless specifically set forth in the claims, the invention
is not limited to implementation using a specific programming
language, operating system environment or hardware platform.
[0108] It is to be understood that the embodiments of the present
invention which have been described are merely illustrative of some
of the applications of the principles of the present invention.
Numerous modifications may be made by those skilled in the art
based upon the teachings presented herein without departing from
the true spirit and scope of the invention. The contents of all
references, patents and published patent applications cited
throughout this application are hereby incorporated by reference in
their entirety for all purposes.
[0109] The following examples are set forth as being representative
of the present invention. These examples are not to be construed as
limiting the scope of the invention as these and other equivalent
embodiments will be apparent in view of the present disclosure,
figures, tables, and accompanying claims.
Example I
Overall Strategy for Oligopaint Design
[0110] 1. Give centromeres an identifying color: e.g., can either
make all centromeres the same color, or make
chromosome-specific.
[0111] 2. Query whether minor M-bands will obscure major M-bands.
Minor:major M-band ratios such as, e.g., 1:1, 2, 3, 4, 5, 10, 20,
50, 100 will be tried.
[0112] 3. Query what distance the M-bands should be from one
another to be distinct. 250 and 500 kb, as well as 1, 2, 3, 4, 5,
6, 7, 8, 9, and 10 Mb will be tried.
[0113] 4. Whether there is a pattern of I- and M-bands (varying
color, thickness) that can uniquely identify regions will be
determined. All combinations of 2, 3, 4, & 5 colors, spaced 50,
100, 200, 500, and 750 kb, and 1 and 2 Mb apart for separation and
color interference will be tested. Five colors in a never-repeating
pattern may permit unambiguous tagging of all genomic regions, but
may raise challenges: a) condensation, which can also be uneven,
may change colors. Varying band widths of a color may avoid issues
because (without intending to be bound by scientific theory)
condensation should not change hue.
[0114] 5. How close (far apart) I-bands must be to make a single
band (be distinct) will be determined. Try 10, 30, 50, 75, 100,
150, 200, 500, 750, and 1000 kb.
[0115] 6. How many x-mers are needed to produce an I-band will be
determined. A contiguous 1, 3, 5, 7, and 10 kb will be labelled for
all colors.
[0116] 7. How long the probe/primer sequences should be will be
determined. Probe lengths of 40-28 bases and corresponding primer
lengths of 10-16 bases will be tested, and this will be done for
varying GC content. Pre-selections will be performed to avoid
overlap with other primers and any unique sequence in the genome.
Also, primers can be extended by adding tails after synthesis of
array. Primers and extensions will be designed such that they do
not overlap unique sequence other primers.
[0117] 8. Watson & Crick strands will be separately labelled.
Different primers will be used for 5' and 3' ends, and N will be
placed on only one of the two.
[0118] 9. Whether universal primers should be used will be
determined. Although universal primers can be used, although their
usefulness is unclear.
[0119] 10. Targets will be pre-selected to minimize partial
homology to repetitive elements to avoid repetitive sequences. If
problems arise after arrays are made, the following steps may be
employed: a) compete with unlabeled probe, b) remove oligo from
library by hybridizing w/i) homologous RNA and removing by
anti-RNA/DNA or ii) homologous DNA-biotin, putting through
column.
[0120] 11. Other avenues: a) Electron dense material will be used
for EM studies, b) bleeding of colors will be used to help study
condensation.
[0121] 12. Give unique colors to: ultraconserved elements (UCEs)
(e.g., intergenic/introic/exonic); imprinted regions; allelically
skewed genes; exons; cell-type (e.g., stem) markers and the
like.
Example II
Restriction-Free Protocol to Make Oligopaint Probes
[0122] The basic idea is to make 60-mers on the Agilent platform:
10 base on each end for the quasi-universal primers and 40 bases in
the middle representing the unique regions of the human genome
(FIG. 5).
[0123] Parameters: [0124] 1. Minimum density 40-mer tiling (half
that, 1/4, 1/8, etc.) [0125] 2. Minimum length: 5 kb (3 kb, 7 kb, 9
kb) [0126] 3. Minimum interphase distance between bands: 40 kb (20,
60, 80) [0127] 4. Minimum metaphase distance between bands: 4 Mb
(2, 6, 8) [0128] 5. Length of primers: 10 bp (9, 12, 14, 15)
[0129] Given a 40:1 compaction ratio for 30 nm chromatin, the
DNA:Interphase(I):Metaphase(M) compaction ratios for chromosome 19
are (64 Mb) 21 mm:500 microns:5 microns=4000:100:1. A microscopic
resolution of 300 nm means 40 kbp/pixel interphase and 4 Mbp/pixel
for metaphase.
[0130] Goals for Color Layout: [0131] 1) Know the location in I or
M with minimal context and counting. [0132] 2) Be suitable for
human or computer reading [0133] 3) Have one set of paints to cover
the interphase to metaphase transition [0134] 4) Be able to
uniformly label by whole chromosome or by arm or by strand. [0135]
5) Be able to selectively amplify from up to 100 chips which don't
necessarily neatly end at the arm boundaries.
[0136] Assuming that 5 kb of solid (unique) 40-mers is enough to
detect as a band in both I & M, 35 kb between bands in I, 4 Mb
in M. Human chromosomes range from 47 Mb=16 M-bands (#21) to 247
Mb=80 bands (#1).
[0137] Each set of 4 M-bands is enough to encode 5 4=625 bands
(enough to cover the 800 such overlapping 16 Mbp regions with some
redundancy considering that the I-bands contribute a 5th). Each
M-band has 100 I-bands. Ten I-bands are enough to encode a unique
(7 bit, 2 7=128) binary pattern, which can be augmented with 3
check bits and repeated 10 times, for example:
[0138] Chromosome 1=4 sets of 4 M-bands--with I-band color in
parenthesis, 50 to 90 bands out of 100 (v. 1 out of 100 for
M-bands): 4(5 . . . )4(5 . . . )4(5 . . . )4(5 . . . )1(5 . . .
)4(5 . . . )4(5 . . . )4(5 . . . )2 (see scenario #1 below).
[0139] Expanding the first of the 16 M-bands below: [0140] Paint:
45555.55.5.5555.55.5.5555.55.5.5555.55.5.5555.55.5 .5555.55.5.45
[0141] 1s digit 01234567890123456789012345678901234567890123456789
0123456789012 [0142] 10s digit
00000000001111111111222222222233333333334444444444 . . .
9999999999000 [0143] 100s digit (Position 0 to 102) 111
[0144] A De Bruijn sequence B(k, n) is a cyclic sequence of a given
alphabet size k for which every possible subsequence of length n
appears as a sequence of consecutive characters exactly once
(length=k n) (See Worldwide Website
hakank.org/comb/deBruijn.Applet.html).
[0145] Scenario #1: Chips are generated in order of position on the
genome, so by labeling one chip out of N, that fraction of the
genome is obtained (this is not going to perfectly coincide with a
chromosome boundary unless a few chips are wasted). Below are five
B(4,4) sequences (5*256 M-bands each) which should be more than
enough for encoding the roughly 800 M-bands (depending on optimal
density from the first chip experiment). In each set below the
(missing) 5th color is the (dominant) I-band color. In this case
each color has its own primer pair (5 total). Note that since these
sequences are 256 characters long, they don't fit on the line but
instead wrap to the next line. The first 256-mer below assumes an
I-band color #5:
[0146]
4444144424443441144124413442144224423443144324433414142414341114112-
41134
12141224123413141324133424243421142124213422142224223423142324233434-
3114
312431343214322432343314332433311112111311221123113211331212131222122-
312 3212331313221323133213332222322332323333 [0147]
555515552555355115512551355215522552355315532553351515251535111511251135
121512251235131513251335252535211521252135221522252235231523252335353115
312531353215322532353315332533311112111311221123113211331212131222122312
3212331313221323133213332222322332323333 [0148]
444414442444544114412441544214422442544514452445541414241454111411241154
121412241254151415241554242454211421242154221422242254251425242554545114
512451545214522452545514552455511112111511221125115211551212151222122512
5212551515221525155215552222522552525555 [0149]
444414445444344114415441344514455445344314435443341414541434111411541134
151415541534131413541334545434511451545134551455545534531453545334343114
315431343514355435343314335433311115111311551153113511331515131555155315
3515331313551353133513335555355335353333 [0150]
444424445444344224425442344524455445344324435443342424542434222422542234
252425542534232423542334545434522452545234552455545534532453545334343224
325432343524355435343324335433322225222322552253223522332525232555255325
3525332323552353233523335555355335353333
[0151] Scenario #2: As per Scenario #1 except 24 I-band colors
(A-X)are used, which means that the De Bruijn alphabet (for the
M-bands) can only be k=3 (not 4 colors in Scenario #1) since now
two colors are used just for the I-bands. Below are 24 B(3,4)
sequences (24*81 M-bands each) which should be more than enough for
encoding the roughly 800 M-bands (depending on optimal density from
the first chip experiment). In each set below the (missing) 1 or 2
colors combine to form the (dominant) I-band color (or 24
combinations total). Since each I-band has its one primer pair and
the five primary colors have their own primer pairs (for the
M-bands), in principle anyone could get any combination of
chromosome and color combination and strand simply by how the
primers are labeled (and independent of chip#). This can be easily
extended to all 48 arms by assigning two primer pairs for each of
the 24 color-combinations (one each for p & q arms). Since
these sequences are 27 characters long, they don't fit on the line
and instead wrap to the next line. The first 27-mer below assumes
an I-band color using #4 and 5 or just #4 or just #5.
[0152] I:4&5: 3 3 3 3 1 3 3 3 2 3 3 1 1 3 3 1 2 3 3 2 1 3 3 2 2
3 1 3 1 3 2 3 1 1 1 3 1 1 2 3 1 2 1 3 1 2 2 3 2 3 2 1 1 3 2 1 2 3 2
2 1 3 2 2 2 1 1 1 1 2 1 1 2 2 1 2 1 2 2 2 2 [0153] I:3&5: 4 4 4
4 1 4 4 4 2 4 4 1 1 4 4 1 2 4 4 2 1 4 4 2 2 4 1 4 1 4 2 4 1 1 1 4 1
1 2 4 1 2 1 4 1 2 2 4 2 4 2 1 1 4 2 1 2 4 2 2 1 4 2 2 2 1 1 1 1 2 1
1 2 2 1 2 1 2 2 2 2 [0154] I:3&4: 5 5 5 5 1 5 5 5 2 5 5 1 1 5 5
1 2 5 5 2 1 5 5 2 2 5 1 5 1 5 2 5 1 1 1 5 1 1 2 5 1 2 1 5 1 2 2 5 2
5 2 1 1 5 2 1 2 5 2 2 1 5 2 2 2 1 1 1 1 2 1 1 2 2 1 2 1 2 2 2 2
[0155] I:2&5: 4 4 4 4 1 4 4 4 3 4 4 1 1 4 4 1 3 4 4 3 1 4 4 3 3
4 1 4 1 4 3 4 1 1 1 4 1 1 3 4 1 3 1 4 1 3 3 4 3 4 3 1 1 4 3 1 3 4 3
3 1 4 3 3 3 1 1 1 1 3 1 1 3 3 1 3 1 3 3 3 3 [0156] I:1&5: 4 4 4
4 2 4 4 4 3 4 4 2 2 4 4 2 3 4 4 3 2 4 4 3 3 4 2 4 2 4 3 4 2 2 2 4 2
2 3 4 2 3 2 4 2 3 3 4 3 4 3 2 2 4 3 2 3 4 3 3 2 4 3 3 3 2 2 2 2 3 2
2 3 3 2 3 2 3 3 3 3 [0157] I:1&4: 2 2 2 2 5 2 2 2 3 2 2 5 5 2 2
5 3 2 2 3 5 2 2 3 3 2 5 2 5 2 3 2 5 5 5 2 5 5 3 2 5 3 5 2 5 3 3 2 3
2 3 5 5 2 3 5 3 2 3 3 5 2 3 3 3 5 5 5 5 3 5 5 3 3 5 3 5 3 3 3 3
[0158] I:1&3: 2 2 2 2 5 2 2 2 4 2 2 5 5 2 2 5 4 2 2 4 5 2 2 4 4
2 5 2 5 2 4 2 5 5 5 2 5 5 4 2 5 4 5 2 5 4 4 2 4 2 4 5 5 2 4 5 4 2 4
4 5 2 4 4 4 5 5 5 5 4 5 5 4 4 5 4 5 4 4 4 4 [0159] I:1&2: 4 4 4
4 5 4 4 4 3 4 4 5 5 4 4 5 3 4 4 3 5 4 4 3 3 4 5 4 5 4 3 4 5 5 5 4 5
5 3 4 5 3 5 4 5 3 3 4 3 4 3 5 5 4 3 5 3 4 3 3 5 4 3 3 3 5 5 5 5 3 5
5 3 3 5 3 5 3 3 3 3 [0160] I:2&3: 4 4 4 4 5 4 4 4 1 4 4 5 5 4 4
5 1 4 4 1 5 4 4 1 1 4 5 4 5 4 1 4 5 5 5 4 5 5 1 4 5 1 5 4 5 1 1 4 1
4 1 5 5 4 1 5 1 4 1 1 5 4 1 1 1 5 5 5 5 1 5 5 1 1 5 1 5 1 1 1 1
[0161] I:2&4: 1 1 1 1 5 1 1 1 3 1 1 5 5 1 1 5 3 1 1 3 5 1 1 3 3
1 5 1 5 1 3 1 5 5 5 1 5 5 3 1 5 3 5 1 5 3 3 1 3 1 3 5 5 1 3 5 3 1 3
3 5 1 3 3 3 5 5 5 5 3 5 5 3 3 5 3 5 3 3 3 3
TABLE-US-00001 [0161] TABLE 1 Chromosome # Mb 1 447 4 444 4 400 4
191 5 181 6 171 7 159 8 146 9 140 10 145 11 144 14 144 14 114 14
106 15 100 16 89 17 79 18 76 19 64 40 64 41 47 44 50 44-X 155 44-Y
58 4079
REFERENCES
[0162] Schrock et al. (1996) Science 474(5474):494
[0163] Worldwide Website: ncbi.nlm.nih.gov/pubmed/11044455
[0164] Worldwide Website: ncbi.nlm.nih.gov/pubmed/10479870
[0165] Worldwide Website: ncbi.nlm.nih.gov/pubmed/8664547
[0166] Cross-species color segmenting or RxFISH, barcodes from
fragmented hybrids (Worldwide Website:
chrombios.com/AboutFISH/BarCodes.html)
[0167] Multicolour (44 color) fluorescence in situ hybridisation
(mFISH), multicolour banding analysis (mBAND), region-specific
partial chromosome paints from Metasystems (Germany) (Worldwide
Website: ori.nus.edu.es/MCytogenetics.html)
[0168] Multicolor FICTION, DNA labelling were diethylaminocoumarin
(DEAC), SpectrumGreen.TM. (SG), SpectrumOrange.TM. (SO), Texas
Red.RTM. (TR) and Cyanine 5 (Cy.TM.5), detection of the
immunophenotype was performed with aminomethylcoumarin (AMCA)
(Worldwide Website: metasystems.de/customers/a04/a04.htm)
[0169] All STAR*FISH paint systems for whole human chromosomes
(Worldwide Website:
openbiosystems.com/FISHprobes/Starfish/Human/Multicolor/)
[0170] CTs 4 green (labeled with dinitrophenol, detected with
FITC), CTs 5 blue (labeled with digoxigenin, detected with Cy4),
and CTs 11 red (labeled with biotin, detected with Cy5) (Worldwide
Website:
cshprotocols.cshlp.org/cgi/content/full/4007/10/pdb.prot4740/F4).
Example III
Making Probes Using dU Digestion
[0171] To determine whether USER.TM.-digested, synthesized
oligonucleotides having an internal fluor could be used in FISH, 60
base pair probes were synthesized (as versus PCR amplified),
mimicking what would be expected if the oligonucleotides had been
generated by PCR. The probes contained 32 base pairs of homology to
a locus in Drosophila that contains approximately 110 copies of the
target sequence. Both strands of a 60 base pair oligonucleotide
having internal dUs and internal fluors were synthesized. The two
synthesized oligonucleotides were mixed in equal portions and
cleaved at the dUs with the USER.TM. (uracil-specific excision
reagent) enzyme (New England Biolabs, Ipswich, Mass.). The
oligonucleotides were then used for FISH, with a single-stranded 32
base pair oligonucleotide targeting a different sequence in the
same region as a control.
[0172] It was determined that double stranded, 32 base pair
oligonucleotides could be used as FISH probes, but double stranded,
60 base pair oligonucleotides could not. Since the double stranded
PCR products would be 60 base pairs in length, a strategy was
developed for modifying them prior to FISH. PCR primers that
carried an internal dU and an internal fluor were used such that
the 5' ends of the primers could be excised with USER.TM.
subsequent to PCR (FIG. 7). It was determined that
USER.TM.-digested, synthesized (not PCR amplified) oligonucleotides
could be used in FISH.
[0173] Having determined that the use of internal dUs and internal
fluors permitted synthesized, double stranded, 60 base pair
oligonucleotides to be used as probes, it was next queried whether
analogous PCR generated 60 base pair oligonucleotides could also be
used as probes. It was determined that USER.TM.-digested, PCR
generated, double stranded, 60 base pair oligonucleotides could
indeed be used in FISH.
[0174] Synthesized Oligonucleotides
[0175] USER.TM.-digested, synthesized oligonucleotides were used in
FISH at 100 ng, 200 ng, 400 ng and 800 ng concentrations. 200 ng of
single stranded, 32 base pair oligonucleotide was used as a
control. FISH was performed as follows: 30 minute hybridization at
room temperature, two 10 minute washes, auto leveled using
Photoshop, 60.times. objective, NA=1.2, 1 second exposure.
[0176] PCR Generated Oligonucleotides
[0177] USER.TM.-digested, PCR generated oligonucleotides were used
in FISH at 50 ng, 100 ng, 200 ng and 400 ng concentrations. 200 ng
of single stranded, 32 base pair oligonucleotide was used as a
control. FISH was performed as follows: 30 minute hybridization at
room temperature, two 10 minute washes, auto leveled using
Photoshop, 60.times. objective, NA=1.2, 1 second exposure.
Example IV
Enhancing Signal To Noise
[0178] Many protocols relying on hybridization of nucleic acid
probes to nucleic acid targets aim to optimize signal to noise by
increasing the affinity of the probe to its target and decreasing
the affinity of the probe to background. The following strategies
will be used to increase signal to noise ratios:
[0179] 1. The length of the probe will be extended via
polymerization along a nucleic acid target, e.g., a chromosome,
thereby increasing the affinity of the probe to its target. Without
intending to be bound by scientific theory, probes that are
incorrectly hybridized to targets or non-specifically bound to
non-nucleic acid substrates will not be subject to extension, thus
increasing signal to noise ratios.
[0180] 2. Probes that include one or more quenchers and one or more
fluorescent tags will be used such that when a probe is hybridized
to a nucleic acid target and extended, the quenchers will be
released. Without intending to be bound by scientific theory, this
should enhance signal to noise ratios.
[0181] 3. When hybridizing probes to cells or other complex
targets, the amount of non-nucleic acid substrates present will be
reduced through the use of proteinases, lipases and the like.
Without intending to be bound by scientific theory, this should
enhance signal to noise ratios.
Example V
Oligopaints
[0182] Currently, companies such Open BioSystems and Metasystems
use FACS-sorted chromosomes, which can also be microdissected into
smaller fragments, to generate chromosome paints. This approach can
provide up to 500 colored bands of per haploid genome
(Metasystems), corresponding to approximately 6 Mb of DNA per band.
The price of these paints ranges from approximately $100 to $4,000
per genome per assay, with chromosome paints that provide higher
resolution costing significantly more than whole chromosome
paints.
[0183] The cost of paints can be greatly reduced by synthesizing
them via PCR amplification of oligomers (e.g., 60-mers) that
consist of genomic sequences (e.g., 32-mers) (representing only the
unique part of the genome) flanked by primer sequences (e.g.,
14-mers) and, in total, represent 20% (although the oligomer
lengths and percentages may differ depending on array optimization
experiments, the type of genome, the AT content of the genome,
spacing of repeated sequences with unique sequences, etc.) of the
human genome (FIG. 1). Oligomer sizes described in this paragraph
(e.g., primer sequences and/or genomic sequence) may be increased
or decreased based on the results of optimization experiments.
These 60-mers will be synthesized on Agilent 244K arrays at the
cost of $500 per array such that 20% of the human genome will be
contained on 80 to 95 arrays (FIG. 2). Judicious design and use of
the primer sequences will then, in conjunction with the subdivision
of the genome into 80 to 92 sub-chromosomal arrays, allow for the
separate amplification and labeling of approximately 664 pools of
genomic sequence. Application of all 664 pools of probe will then
constitute a whole genome chromosome paint which, without intending
to be bound by scientific theory, will produce a crisp banding
pattern on metaphase chromosomes and increasingly finer banding
patterns on increasingly decondensed chromosomes (FIG. 3).
Importantly, after the initial expense of the arrays, the cost of
maintaining the templates by PCR for the future batches of paints
will be minimal, dropping the cost of the paints to dollars per
assay (including the cost of primers and dyes). Each step of this
protocol has been carried out successfully.
[0184] The Oligopaints and methods of making them described herein
provide numerous advantages over chromosome paints that are
commercially available. For example, Oligopaints and methods of
making them provide: 1) increased resolution over chromosome paints
that are commercially available; 2) reduced price over chromosome
paints that are commercially available; 3) availability for any
organism for which there is a genome sequence, even if that
sequence is partial (that is, YACs, BACs and/or chromosomes that
are sortable (e.g., by FACS) are not necessary); 4) the ability to
avoid background issues caused by repetitive sequences, because the
use of repetitive sequences can be avoided (in contrast, chromosome
paints that are commercially available use "cold" (i.e., unlabeled)
repeat sequences to outcompete the labeled probes; 5) the option to
eliminate certain bandings by not amplifying probes to those bands
(e.g., after an array has been generated); 6) the option to
redesign arrays to fit individual needs; and 7) the ability to
specifically label certain sequences by giving them identifying
primer sequences.
[0185] The Oligopaints described herein are useful for a variety of
methods including, but not limited to: 1) heterologous and/or
homologous (e.g., pairing) interchromosomal interaction studies; 2)
intrachromosomal organizational studies such as, e.g., looping,
coiling and the like; 3) chromosome organizational studies, such
as, e.g., chromosome path, placement of specific sequences and the
like; 4) chromosome condensation studies, such as, e.g., mitosis,
meiosis, arrest during cell cycle and the like (new colors will be
generated when Oligopainted bands overlap and/or `bleed` into one
another); 5) chromosome behavior studies such as, e.g.,
segregation, motion in non-dividing cells and the like; 6)
karyotyping studies such as, e.g., for medical science (e.g.,
diagnostic karyotyping, amniocentesis, pre-implantation diagnosis
and the like), for basic science, to detect copy number variations
(CNVs) and other chromosomal rearrangements, changes in ploidy and
the like; 7) replication studies such as, e.g., timing,
organization, Bell nuclei and the like; 8) chromosome structure
studies such as, e.g., organization at the electron microscopy
level and the like; and 9) strand specific biology of DNA through
separate labeling of each of the two strands of a DNA double
helix).
[0186] FIG. 1 illustrates how a new form of chromosome paints,
Oligopaints, can be made from template oligonucleotides which are
synthesized on arrays (e.g., chips). Primers are annealed to
sequences on the array and then extended to generate a 60-mer
products which could then be dissociated from the array and used
for second strand synthesis. Products are then aliquoted into
smaller pools which could be amplified with a single primer pair
each. Finally, chromosome paints are made by amplifying the pools
with primers containing two or more dU nucleotides and a
fluorescent dye at the 3' end, followed by cleavage at the dUs to
reduce inter-primer annealing. Note that if the two members of a
primer pair are different in sequence, it will be possible to
differentially label the two strands of DNA, enabling
strand-specific hybridization to DNA or RNA. Note that the first
few steps could be simplified if the original synthesized
oligonucleotide can be released from the array. In certain
exemplary embodiments, the use of dU allows for cleavage of primer
sequences (at the dU), which will reduce the concentration of
primer sequences present during hybridization.
[0187] FIG. 2 schematically depicts how the 24 chromosomes of the
human genome could be differentially colored with a base color for
the interphase bands being a mix of five primary colors, and a
series of color-coded metaphase bands at staggered positions along
the p and q arms. These patterns will be generated by computer
algorithms that select unique sequences along the chromosomes and
then associate them with primer sequences such that their
amplification with corresponding oligos carrying the correct
balance of dyes, followed by hybridization to the chromosome,
which, without intending to be bound by scientific theory, will
likely create identifying banding patterns for sub-chromosomal
regions, especially when the chromosome is decondensed.
[0188] FIG. 1 shows how a band that appears thick on a metaphase
chromosome will likely disperse into a pattern of thinner bands,
which it is hoped will demarcate specific chromosomal regions on
the order of 10-50 kb in size. Currently, arrays are being
synthesized to a) confirm preliminary data demonstrating that 14
base pair primers are sufficiently robust for use in PCR
amplification, b) determine how many oligonucleotide probes are
necessary to generate a visible band in interphase and/or
metaphase, c) determine how far apart oligonucleotide probes must
be to generate distinct bands in interphase and/or metaphase, d)
assess what level of variation there may be in banding patterns
from one chromosomal region to another, e) determine what level of
interference there may be when fluorescent dyes are tightly packed,
and f) ascertain whether the interference can be taken advantage of
to assess degrees of chromosome condensation. Analogous arrays for
the Drosophila and C. elegans genomes are also being designed for
ongoing projects studying pairing in these organisms.
[0189] One innovative aspect of the oligopainting methods and
compositions described herein is the use of computationally
patterned synthetic probes and arrays (rather than natural
DNAs/chromosomes) to generate chromosome paints. This strategy will
enable huge improvements in cost and resolution.
Example VI
Homology Effects
[0190] Oligopaint technology will enable the systematic
investigation of tumor cells and cancer cell lines in terms of
their chromosome arrangement and positioning and, in doing so, will
both emphasize experiments that are often not routinely considered
in terms of cancer as well as demonstrate an affordable resource
that will make such experiments generally feasible. Oligopaint
technology will also enable the search for genes involved in
somatic homolog pairing by permitting whole genome FISH-based
screens of the human genome using Oligopaints in the format of
384-well plates. It was determined that FISH-based screens in
384-well plates was successful. However, many in the art have
predicted that such an approach would not be technically and/or
practically feasible for whole genome screens, especially if the
FISH were to target unique sequences. Oligopaints will make this
approach both technically and practically feasible. As a
whole-genome Oligopaint FISH-based strategy offers a new for
approach for identifying genes that affect chromosome organization,
it will open up new lines of investigation. In particular, attempts
will be made to identify genes that promote homolog pairing, as
such genes could be used to enhance gene replacement and gene
therapy strategies that rely on homologous recombination.
[0191] Human chromosome karyotyping is a routine procedure for the
analysis of cancer genotypes as well as many genetic diseases, such
those associated with a multitude of birth defects associated with
whole chromosome anueploidies, deletions, duplications,
translocations and inversions. Furthermore, with the increased
awareness of copy number variation and the association of such
chromosomal structures with disease, the demand for karyotyping
grows along with the need for increased accuracy.
[0192] Chromosome painting improves the power of karyotyping by
color-coding chromosomes and sub-chromosomal segments. The ability
of physicians to visualize the underlying chromosomal basis for
disease is key for accurate diagnosis and treatment, making the
availability of affordable painting techniques a top priority in
the medical innovation.
[0193] Better painting technologies will also impact the fields of
genetic counseling and prenatal diagnosis. Here, the accuracy of
karyotyping is frequently the determining factor in the quality of
information physicians and genetics counselors can offer patient
clients seeking explanations for their ailments or wishing for a
deeper understanding of the genotypes they have inherited and may
pass on to their children. Unlike the karyotyping of patients whose
disease syndromes will often have already suggested likely
chromosomal abnormalities, clients seeking genetic counseling or
prenatal diagnoses often seek information without any underlying
syndromes. In these situations, the accuracy of the analyses will
rest to a great extent on the resolution of chromosome paints
across the entire genome and, therefore, the higher the resolution
of the paints, the more reliable will be the information obtained.
Unfortunately, high resolution paints can cost thousands of dollars
for a single assay of an entire human genome. One goal is to
produce chromosome paints of the highest resolution for a fraction
of the cost of current high resolution paints. As such, resources
will be available to all populations, especially those for whom
medical services are already an excessive financial burden.
[0194] Finally, the methods and compositions described in herein
should affect a broad spectrum of research fields, including those
focusing on chromosome organization, chromatin structure,
interchromosomal interactions, chromosome transmission, homology
effects, replication, homologous recombination, genome integrity,
and genome evolution. These fields center to a great extent on the
concept of the chromosome as an entity in and of itself, something
more than a repository of genes. As chromosomes are difficult to
study in their entirety except when examined in situ, protocols for
visualizing them are of utmost importance.
[0195] Using techniques ranging from traditional genetics to FISH,
3C analysis, 4C analysis, 5C analysis and microarrays, researchers
are cataloguing hundreds of interchromosomal interactions, some
being specific between two loci and others arising from the
clustering of loci at transcription factories or other nuclear
structures. In short, the popular view of a gene, with enhancers
and promoters arrayed along a single black line, has been found
lacking The oligopainting methods and compositions described herein
will enable one of skill in the art to focus on homology effects.
Homology effects encompass the many forms of gene regulation that
are sensitive to, or reflect, the presence of homology within a
nucleus. The most celebrated of these would include three processes
that occur in humans and other mammals: X-inactivation, where one
of two X chromosomes is inactivated (Bacher et al. (2006) Science
311:1149; Xu et al (2006) Science 311:1149, Epub 2006 Jan. 19))
monoallelism (Borst (2002) Cell 109(1):5; Yang et al. (2007) Cell
128:777), where only one allele of a gene is expressed, and
parental imprinting, a form of monoallelism that reflects the
parental origin of each allele (Edwards et al. (2007) Curr. Opin.
Cell Biol. 19:281; Pauler et al. (2007) Trends Genet. 23:284).
Homology effects are also abundant in fungi, insects, worms, and
plants. For example, a mere 450 base pairs of homology introduced
by a transgene into the fungus, Neurospora, will trigger C to T
mutations within the duplicated regions (Selker (2004) Cold Spring
Harb. Symp. Quant. Biol. 69:119), while 90 base pairs of homology
between a transgene and the tobacco genome will cause methylation
and silencing (Matzke and Matzke (2004) PLoS Biol. 2:E133). These
phenomena demonstrate an uncanny ability of organisms to respond to
homology and, as these responses to homology affect gene
regulation, homology effects are of great relevance to human
development and health.
[0196] Some homology effects are brought about through physical
pairing of the interacting homologous genes and/or chromosomal
regions. Examples of these types of homology effects are now known
to occur in a wide variety of species, including humans and other
mammals, insects and fungi. Among the most dramatic in mammals
would be X-inactivation, where pairing of the X-inactivation center
plays a role in the counting of X chromosomes and the subsequent
process of inactivation. Mammals, like Neurospora, also sport a
process call meiotic silencing of unpaired DNA/chromatin (MSUD/C)
(Turner (2007) Development 134:1823), wherein regions of the genome
that remain unpaired in meiosis are silenced. Without intending to
be bound by scientific theory, this process may explain the curious
phenomenon of meiotic sex chromosome silencing, which occurs in
male meiosis and targets the unpaired regions of the X and Y
chromosomes.
[0197] It has been determined that pairing can cause enhancers of a
gene to act in trans on the promoter of another gene lying on a
separate chromosome, and the cis-trans choice of an enhancer can be
controlled by the integrity of the promoter lying in cis to the
enhancer. Pairing of an internally deleted gene with a homolog
bearing an insulator can lead to changes in gene topology which
allow bypass of the insulator (Morris et al. (1998) Proc. Natl.
Acad. Sci. USA 95:10740). These two mechanisms of pairing-mediated
changes in gene regulation argue that somatic homolog pairing is a
potent form of gene regulation that warrants analysis in any
diploid organism, including humans.
[0198] The oligopainting methods and compositions described herein
can be used to identify factors that mediate homolog pairing and,
to this end, genetic screens have been conducted in Drosophila.
While such an approach has pointed to a handful of candidate genes
involved in gene regulation and chromosome structure (e.g., Hartl
et al. (2008) Science 322:1384; Williams et al. (2007) Genetics
177:31), progress has been slow because past genetic screens have
had to rely on observations of pairing-sensitive phenotypes, which
are sufficiently removed from the process of pairing that they can
complicate analyses. These screens have also been hindered by the
need for organismal viability and the multi-tissue nature of the
whole organism, which prohibits finer levels of structural
analyses. For these reasons, much effort has been exerted to
establish a Drosophila cell culture system for the analysis of
pairing via FISH assays. Cell culture provides homogeneous
populations of cells for biochemical and molecular biological
analyses (Ashe et al. (1997) Genes Dev. 11:2494).
[0199] A protocol permitting FISH assays in the 384-well format was
developed. Using this protocol, sub-genome pilot runs surveyed 11%
of the RNAi library representing the Drosophila genome, yielding a
handful of candidate genes (FIG. 6). These runs addressed two
important points. First, they documented the feasibility of FISH in
the 384-well format. Second, they demonstrated the capacity of
computerized image analysis to detect changes in the pattern of
FISH signals from well to well. As discussed further below, this
protocol will be adapted for use with human cells.
[0200] The biology of pairing will be studied, and studies will
begin with a survey of human transformed cells taken directly from
a wide variety of tumors as well as cell lines. FISH will be
applied using whole genome Oligopaint methods and compounds
described herein and to determine the state of pairing along the
length of each chromosome arm, taking advantage of computer-based
imaging techniques to allow the examination of individual
chromosomes or any combination of thereof.
[0201] To detect low levels of pairing or pairing that may be
specific for certain phases of the cell cycle, at least 100 cells
per arm will be scored, and whether the cells appear to be entering
mitosis or not will be recorded. Because the degree of proximity
may vary along a chromosome arm, measurements of inter-homolog
distance will be made at multiple positions along each arm,
especially for the longer arms, by taking advantage of bar coding
implemented for Oligopaints.
[0202] The methods for measuring pairing will depend to a great
extent on the resolution of the chromosome paints and whether and
how well they will permit the examination of decondensed
chromosomes. Based in part on the methods and compositions
described herein that provide Oligopaints at a very low cost,
future experiments will not be limited in terms of probe and,
therefore, a survey that is far-reaching and comprehensive will be
able to be conducted.
[0203] Normal cells from a wide variety of tissue types will also
be examined, as pairing has never been systematically assessed for
humans or any mammal. Of the few studies looking for somatic
pairing via FISH, probes have generally targeted only single loci
on single chromosome arms and, without intending to be bound by
scientific theory, it remains possible that somatic pairing is more
frequent than is currently predicted. In contrast, studies will be
performed with chromosome paints for all the chromosomes. Together
with an analysis of transformed cells, these studies of normal
cells will determine whether somatic pairing is a common feature of
human cells. Whether pairing is found only in renal oncocytomas
(Koeman et al. (2008) PLoS Genetics 4:e1000176) or in other
transformed cells as well will be studied. If pairing is found
outside of renal oncocytomas, it will be studied whether it is
restricted to transformed cells or whether it can occur in other
types of diseased tissues. Finally, if pairing can be found at a
reasonable level in human cells, it will be studied whether it is
restricted to only certain chromosomes. These are the important
questions that can only be answered by a broad, comprehensive, and
unbiased sampling of cell types.
[0204] One advantage of whole genome chromosome painting over other
technologies is that it will permit the analysis of
interchromosomal interactions at the single cell level. This
resolution will address questions about cell-to-cell variation as
well as correlations between different patterns of interchromosomal
interactions that might be obscured when assays are done on large
populations of cells. In short, although the methods and
compositions described herein focus on pairing, Oligopaints will
allow the analysis of other phenomena as well. The survey of tumor
and normal cells will be an important undertaking As pairing is a
powerful modulator of gene expression, it is important, regardless
of the outcome, to determine the level at which it occurs in human
cells.
[0205] Experiments to identify genes in the human genome that are
involved in homolog pairing will be performed by conducting a
whole-genome RNAi-driven screen using FISH and chromosome paints to
determine the state of pairing on Chromosome 19. The pairing
extends the entire length of the q arm of Chromosome 19, from
centromere to telomere. Although the q arm is maximally paired, the
p arm remains entirely unpaired. Further, pairing does not extend
to any of the several other chromosomes thus far examined. These
three features of the pairing suggest an arm-based, rather than a
locus-specific or whole-genome, mechanism for pairing. Without
intending to be bound by scientific theory, one possibility for
these observations is that a Chromosome 19 q-arm-specific pairing
mechanism has been induced in renal oncocytomas cell lines.
Alternatively, without intending to be bound by scientific theory,
pairing of the q arm in renal oncocytomas may result from the
release of a mechanism that normally suppresses pairing. Either
way, it appears that pairing is a characteristic feature of renal
oncocytomas brought about by a change that is inherited from
cell-to-cell.
[0206] The gene or genes responsible for the pairing in renal
oncocytomas will be identified through a whole-genome RNAi-driven
screen using FISH and chromosome paints as the phenotypic assay.
Following a modified version of the whole-genome, RNAi-driven,
FISH-based protocol that was applied to the Drosophila genome,
cells will be grown in 384 well plates, and they will be targeted
with the repertoire of RNAi directed against approximately 20,000
human genes provided by the Institute for Chemistry and Cell
Biology (ICCB) screening facility at Harvard Medical School. The
impact of the RNAi will be assessed by visualizing the cells with
FISH and chromosome paints targeting Chromosome 19, wherein the p
and q arms will be differentially labeled. Using computer-aided
analyses, RNAi species that promote pairing of the q arm not the p
arm will be searched for, although any pattern of pairing among the
two arms will also be of interest. All candidates will be confirmed
through additional runs of RNAi, after which the genes identified
by the most effective RNAi species will be characterized through
standard genetic, molecular biological and biochemical studies for
their role in chromosome pairing as well as tumorigenesis.
[0207] The cost of the probe for the screen described above will
cost only approximately $1,200 using the Oligopaint methods and
compositions described herein for Chromosome 19. This would be in
sharp contrast to costs of between $90,000 (Metasystems) and
$700,000 (Open Biosystems) or as much as $1,400,000 (Metasystems),
if the resolution of paints to be purchased were to match that of
the Oligopaints that will be synthesized for Chromosome 19.
[0208] The screen can also be carried out with Oligopaints to the
entire genome so that the impact of RNAi species on the pairing of
all chromosome arms can be displayed simultaneously. Oligopaint
costs for such a global screen would be approximately $28,000. This
global approach may be attempted or, alternatively, an approach
that simultaneously targets several, but not all, of the
chromosomes may be undertaken. Along these lines, a few technical
modifications may be necessary in the adaptation of the Drosophila
screening protocol to the protocol above. In particular, the
Drosophila screen used short oligonucleotide probes containing
locked nucleic acids (LNAs), which allowed a more facile adaptation
of the FISH protocol to the 384-well plate format. In addition, or
alternatively, Oligopaints may incorporate LNAs. If this route is
taken, it may reduce the `density` of probes in the paints in order
to offset the greater cost of LNAs as compared to that of
unmodified bases. In the unlikely case that neither of these
approaches is successful, a screen of the human genome will be
performed using a few LNA probes along Chromosome 19, following,
exactly, the protocol that was used for screening the Drosophila
genome.
REFERENCES
[0209] Koeman J M, Russell R C, Tan M H, Petillo D, Westphal M, . .
. Furge K A. Somatic pairing of chromosome 19 in renal oncocytoma
is associated with deregulated EGLN2-mediated [corrected]
oxygen-sensing response PLoS Genet. 2008 July; 4(7)
[0210] Osborne C S, Chakalova L, Mitchell J A, Horton A, Wood A L,
Bolland D J, Corcoran A E, Fraser P. Myc dynamically and
preferentially relocates to a transcription factory occupied by
Igh. PLoS Biol. 2007 August; 5(8):e192.
[0211] Cooper G M, Nickerson D A, Eichler E E. Mutational and
selective effects on copy-number variants in the human genome. Nat
Genet. 2007 July; 39(7 Suppl):522-9.
[0212] Matsuda K, Tanaka M, Araki S, Yanagisawa R, Yamauchi K,
Koike K. Crypticinsertion into 11q23 of MLLT10 not involved in
t(1;15;11;10)(p36;q11;q23;q24) in infant acute biphenotypic
leukemia. Cancer Genet. Cytogenet. 2009 Apr. 15; 190(2):113-20
[0213] Spilianakis, C. G., M. D. Lalioti, T. Town, G. R. Lee and R.
A. Flavell, 2005 Interchromosomal associations between
alternatively expressed loci. Nature 435: 637-645.
[0214] Ling, J. Q., T. Li, J. F. Hu, T. H. Vu, H. L. Chen et al.,
2006 CTCF mediates interchromosomal colocalization between Igf2/H19
and Wsb1/Nf1. Science 312: 269-272.
[0215] Bacher C P, Guggiari M, Brors B, Augui S, Clerc P, Avner P,
Eils R, Heard E. 2006. Transient colocalization of X-inactivation
centres accompanies the initiation of X inactivation. Nat. Cell
Biol. 8:293-9. Epub 2006 Jan. 24.
[0216] Xu N, Tsai C L, Lee J T. 2006. Transient homologous
chromosome pairing marks the onset of X inactivation. Science.
311:1149-52. Epub 2006 Jan. 19.
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