U.S. patent application number 11/026280 was filed with the patent office on 2005-09-22 for methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids.
Invention is credited to Dafforn, Geoffrey A., Kurn, Nurith.
Application Number | 20050208538 11/026280 |
Document ID | / |
Family ID | 34748895 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050208538 |
Kind Code |
A1 |
Kurn, Nurith ; et
al. |
September 22, 2005 |
Methods for analysis of nucleic acid methylation status and methods
for fragmentation, labeling and immobilization of nucleic acids
Abstract
The invention relates to methods for analysis of nucleic acid
methylation status, and fragmentation and/or labeling and/or
immobilization of nucleic acids. More particularly, the invention
relates to methods for fragmentation and/or labeling and/or
immobilization of nucleic acids comprising labeling and/or cleavage
and/or immobilization at abasic sites.
Inventors: |
Kurn, Nurith; (Palo Alto,
CA) ; Dafforn, Geoffrey A.; (Los Altos, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
34748895 |
Appl. No.: |
11/026280 |
Filed: |
December 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60533381 |
Dec 29, 2003 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6827 20130101; C12Q 2521/531 20130101; C12Q 2525/119
20130101; C12P 19/34 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
We claim:
1. A method for fragmenting a polynucleotide comprising a
methylated nucleotide, said method comprising: (a) cleaving a base
portion of the methylated nucleotide with an agent capable of
cleaving the base portion of the methylated nucleotide, whereby an
abasic site is generated; and (b) cleaving the phosphodiester
backbone of the polynucleotide comprising the abasic site at the
abasic site, whereby polynucleotide fragments are generated.
2. A method according to claim 1, wherein the agent capable of
cleaving the base portion of the methylated nucleotide is selected
from the group consisting of an enzyme, a chemical agent, and
acidic conditions.
3. A method according to claim 1, wherein cleavage of the
phosphodiester backbone is performed with an agent selected from
the group consisting of an enzyme, a chemical agent, acidic
conditions, basic conditions, and heat.
4. A method for labeling a polynucleotide comprising a methylated
nucleotide, said method comprising fragmenting the polynucleotide
according to the method of claim 1, and further comprising: (c)
labeling at the abasic site, whereby a labeled polynucleotide
fragment is generated.
5. A method according to claim 4, wherein the labeled
polynucleotide fragment comprises a detectable label.
6. A method according to claim 5, further comprising hybridizing
the labeled polynucleotide fragment to a probe.
7. A method for labeling a polynucleotide comprising a methylated
nucleotide, said method comprising: (a) cleaving a base portion of
the methylated nucleotide with an agent capable of cleaving the
base portion of the methylated nucleotide, whereby an abasic site
is generated; and (b) labeling at the abasic site, whereby a
labeled polynucleotide is generated;
8. A method according to claim 7, further comprising hybridizing
the labeled polynucleotide to a probe.
9. A method for characterizing a methylated polynucleotide, said
method comprising detecting a polynucleotide fragment, wherein said
polynucleotide fragment is prepared according to the method of
claim 1, wherein detection of said polynucleotide fragment
correlates with presence, absence, sequence, or amount of the
methylated polynucleotide.
10. A method for characterizing a methylated polynucleotide, said
method comprising detecting a labeled polynucleotide, wherein said
labeled polynucleotide is prepared according to the method of claim
7, wherein detection of said labeled polynucleotide correlates with
presence, absence, sequence, or amount of the methylated
polynucleotide.
11. A method for fragmenting a polynucleotide comprising a
methylated nucleotide, said method comprising: (a) cleaving a base
portion of an unmethylated nucleotide with an agent capable of
cleaving the base portion of the unmethylated nucleotide, whereby
an abasic site is generated, wherein the agent is not capable of
cleaving a methylated nucleotide; (b) cleaving the phosphodiester
backbone of the polynucleotide comprising the abasic site at the
abasic site, whereby polynucleotide fragments are generated.
12. A method according to claim 11, wherein the agent capable of
cleaving a base portion of an unmethylated nucleotide comprises an
enzyme.
13. A method according to claim 12, wherein said the unmethylated
nucleotide is cytosine and the enzyme comprises cytosine deaminase
in conjunction with uracil DNA glycosylase.
14. A method according to claim 11, wherein cleavage of the
phosphodiester backbone is performed with an agent selected from
the group consisting of an enzyme, a chemical agent, acidic
conditions, basic conditions, and heat.
15. A method according to claim 11, wherein the polynucleotide
comprising a methylated nucleotide is contacted with a methyl
binding agent prior to or during step (a).
16. A method for labeling a polynucleotide comprising a methylated
nucleotide, said method comprising fragmenting the polynucleotide
according to the method of claim 11, and further comprising: (c)
labeling at the abasic site, whereby a labeled polynucleotide
fragment is generated.
17. A method according to claim 16, wherein the labeled
polynucleotide fragment comprises a detectable label.
18. A method according to claim 17, further comprising hybridizing
the labeled polynucleotide fragment to a probe.
19. A method for labeling a polynucleotide comprising a methylated
nucleotide, said method comprising: (a) cleaving a base portion of
an unmethylated nucleotide with an agent capable of cleaving the
base portion of the unmethylated nucleotide, whereby an abasic site
is generated, wherein the agent is not capable of cleaving a
methylated nucleotide; and (b) labeling at the abasic site, whereby
a labeled polynucleotide fragment is generated.
20. A method according to claim 19, comprising hybridizing the
labeled polynucleotide to a probe.
21. A method for fragmenting and labeling a polynucleotide
comprising a canonical nucleotide, said method comprising: (a)
cleaving a base portion of a canonical nucleotide present in a
polynucleotide comprising the canonical nucleotide with an agent
capable of cleaving a base portion of the canonical nucleotide,
whereby an abasic site is generated; (b) cleaving the
phosphodiester backbone at the abasic site; and (c) labeling at the
abasic site, whereby a labeled polynucleotide fragment is
generated.
22. A method according to claim 21, wherein the agent capable of
cleaving a base portion of a canonical nucleotide comprises an
enzyme.
23. A method according to claim 22, wherein said canonical
nucleotide is cytosine and said enzyme comprises cytosine deaminase
in conjunction with uracil DNA glycosylase.
24. A method according to 21, wherein cleavage of the
phosphodiester backbone is performed with an agent selected from
the group consisting of an enzyme, a chemical agent, acidic
conditions, basic conditions, and heat.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/533,381, filed on Dec. 29, 2003, the disclosure
of which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The invention relates to methods for analysis of nucleic
acid methylation status, and methods for fragmentation and/or
labeling and/or immobilization of nucleic acids. More particularly,
the invention relates to methods for fragmentation and/or labeling
and/or immobilization of nucleic acids comprising labeling and/or
cleavage and/or immobilization at abasic sites.
BACKGROUND
[0003] Methylation of DNA is involved in both normal and abnormal
cellular processes. For example, DNA methylation has been
implicated in X-inactivation, genomic imprinting, and differential
gene expression (such as by upregulation or silencing of genetic
loci). DNA methylation plays a role in gene inactivation, cell
differentiation, tumorigenesis, X-chromosome inactivation, and is
required for mammalian development (Li, et al., Cell 69:915-926,
1992; Okano et al., Cell 99:247-57, 1999). In bacteria, methylation
of cytosine and adenine residues plays a role in the regulation of
DNA replication and DNA repair. DNA methylation has also been
associated with increased risk of cancer, as well as cancer
development itself.
[0004] Methylation of DNA is carried out by methylases (also known
as methyltransferases). These enzymes are generally
sequence-specific, and they can methylate both nucleic acid strands
(in the case of DNA). Replication of these strands yields a
hemi-methylated state which is recognized by a class of maintenance
methylases capable of restoring full methylation to both
strands.
[0005] Methylation can occur at all nucleotide residues, although
in mammalian species, DNA methylation commonly occurs at cytosine
residues, and more commonly at cytosine residues that lie next to a
guanosine residue, i.e., at cytosine residues of a CG dinucleotide.
CG dinucleotides in "CpG islands" remain methylation-free. CpG
islands are rich in CG sites and are often found near coding
regions within the genome (i.e., genes). About half of the genes in
the human genome are associated with CpG islands. Importantly, the
vast majority of CpG islands in the genome remain unmethylated in
normal adult cells and tissues. Methylation of CpG islands is
normally seen only on the inactive X-chromosome in females and at
imprinted genes where it functions in the stable silencing of such
genes. Strict control over the levels and distribution of DNA
methylation are essential to normal animal development.
[0006] Alteration in DNA methylation is one manifestation of the
genome instability characteristic of human tumors. A hallmark of
human carcinogenesis is the loss of normal constraints on cell
growth resulting from genetic alterations in the genes that control
cell growth. The consequences of such mutations include the
activation of positive growth signals and the inactivation of
growth inhibitory signals. Identification of gene targets which
when methylated lead to the loss of normal cell responses would be
valuable. This would facilitate the diagnosis and treatment of
disorders associated with abnormal methylation and any downstream
events resulting therefrom.
[0007] The level of methylation of a nucleic acid can be determined
using a number of techniques available in the art. Some methods of
analysis involve the use of the chemical regent, bisulfite. Other
methods for methylation analysis include methylation-sensitive
restriction analysis, methylation-specific polymerase chain
reaction (MSP), sequencing of bisulfite-modified DNA, Ms-SnuPE, and
COBRA.
[0008] There is a serious need for improved methods for analyzing
nucleic acid methylation status.
[0009] Fragmentation and labeling of nucleic acids are important
for the analysis of nucleic acid sequences. For example,
fragmentation and/or labeling are commonly required for detection
of sequences by binding of a sample nucleic acid to complementary
sequences immobilized on a surface, for example, on a microarray.
Cleavage of sample nucleic acid into small fragments (e.g., 50-100
base pairs) facilitates diffusion of nucleic acid onto the surface,
and may facilitate hybridization. It is known, for example, that
steric and charge hindrance effects increase with the size of
nucleic acids that are hybridized. Moreover, cleavage of sample
nucleic acids into small fragments may ensure that two sequences of
interest in the sample do not appear to bind to the same template
nucleic acid simply by virtue of their proximity on the test
nucleic acid. Cleavage of nucleic acids also facilitates detection
of hybridized nucleic acid when, as in many detection methods, the
size of the signal is proportional to the size of the bound
fragment and thus, control of fragment size is desirable. Labeling
of nucleic acids is necessary in many methods of nucleic acid
analysis because there are presently few techniques for direct
detection of unlabeled nucleic acid with the requisite sensitivity
for analysis on chips. Methods for fragmenting and/or labeling
nucleic acids are known in the art. See, e.g., U.S. Pat. Nos.
5,082,830; 4,996,143; 5,688,648; 6,326,142; and PCT Publication No.
WO 02/090584, and references cited therein.
[0010] Immobilization of nucleic acids to create, for example,
microarrays or tagged analytes, is useful for, e.g., detection and
analysis of nucleic acids and tagged analytes. Methods for
immobilizing nucleic acids are known in the art. See, e.g., U.S.
Pat. Nos. 5,667,979; 6,077,674; 6,280,935; and references cited
therein.
[0011] There is a serious need for improved methods for labeling
and/or fragmenting and/or immobilizing nucleic acids to a surface
(such as a microarray).
[0012] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0013] In one aspect, the invention provides methods for labeling
and fragmenting a polynucleotide comprising a methylated nucleotide
comprising: (a) contacting a polynucleotide comprising (in some
embodiments, suspected of comprising) a methylated nucleotide with
an agent capable of cleaving a base portion of the methylated
nucleotide (i.e., cleaving a base portion of the methylated
nucleotide), whereby an abasic site is generated; (b) cleaving the
backbone of the polynucleotide comprising the abasic site at the
abasic site; and (c) contacting the polynucleotide comprising the
abasic site with an agent capable of labeling the abasic site
(i.e., labeling the abasic site), whereby labeled polynucleotide
fragments are generated. In some embodiments, the agent capable of
cleaving the base portion of the methylated nucleotide is selected
from the group consisting of an enzyme, a chemical agent, and
acidic conditions. In some embodiments, cleavage of the
phosphodiester backbone of a polynucleotide is performed with an
agent selected from the group consisting of an enzyme, a chemical
agent, acidic conditions, basic conditions, and heat. Two or more
of the steps described above may be performed simultaneously, or
the steps may be performed sequentially. For example, steps (a),
(b), and (c) may be performed simultaneously, steps (a) and (b) may
be performed simultaneously, or steps (b) and (c) may be performed
simultaneously. When the steps are performed sequentially, step (b)
may be performed before step (c) or step (c) may be performed
before step (b).
[0014] In another aspect, the invention provides methods for
labeling a polynucleotide comprising a methylated nucleotide,
comprising: (a) cleaving a base portion of the methylated
nucleotide with an agent capable of cleaving the base portion of
the methylated nucleotide, whereby an abasic site is generated; and
(b) labeling at the abasic site, whereby a labeled polynucleotide
is generated.
[0015] In another aspect, the invention provides a method for
fragmenting a polynucleotide comprising a methylated nucleotide,
comprising: (a) cleaving a base portion of the methylated
nucleotide with an agent capable of cleaving the base portion of
the methylated nucleotide whereby an abasic site is generated; and
(b) cleaving the backbone of the polynucleotide comprising the
abasic site at the abasic site, whereby polynucleotide fragments
are generated.
[0016] In another aspect, the invention provides methods for
fragmenting and labeling a polynucleotide comprising a methylated
nucleotide, comprising: (a) incubating a reaction mixture, said
reaction mixture comprising: (i) a polynucleotide comprising a
methylated nucleotide; and (ii) an agent capable of specifically
cleaving a base portion of a methylated nucleotide; wherein the
incubation is under conditions that permit cleavage of the base
portion of the methylated nucleotide, whereby a polynucleotide
comprising an abasic site is generated; (b) incubating a reaction
mixture, said reaction mixture comprising: (i) a polynucleotide
comprising an abasic site; and (ii) an agent capable of effecting
(generally, specific) cleavage of a phosphodiester backbone at the
abasic site; wherein the incubation is under conditions that permit
cleavage of the phosphodiester backbone at the abasic site; whereby
fragments of the polynucleotide are generated; (c) incubating a
reaction mixture, said reaction mixture comprising: (i) a
polynucleotide comprising an abasic site; and (ii) an agent capable
of labeling the abasic site; wherein the incubation is under
conditions that permit labeling at the abasic site; whereby labeled
fragments are generated.
[0017] In one embodiment, the invention provides a method for
fragmenting a polynucleotide comprising a methylated nucleotide,
said method comprising cleaving the phosphodiester backbone of a
polynucleotide comprising an abasic site at the abasic site,
wherein the abasic site is generated by cleaving a base portion of
a methylated nucleotide with an agent capable of cleaving the base
portion of the methylated nucleotide, whereby an abasic site is
generated. In another embodiment, the invention provides a method
for fragmenting and labeling a polynucleotide comprising a
methylated nucleotide, said method comprising cleaving the
phosphodiester backbone of a polynucleotide comprising an abasic
site at the abasic site, wherein the abasic site is generated by
cleaving a base portion of a methylated nucleotide with an agent
capable of cleaving the base portion of the methylated nucleotide,
and labeling at the abasic site, whereby a labeled polynucleotide
fragment is generated. In another embodiment, the invention
provides a method for labeling a polynucleotide comprising a
methylated nucleotide, said method comprising labeling at the
abasic site of a polynucleotide comprising an abasic site, whereby
a labeled polynucleotide is generated, wherein the abasic site is
generated by cleaving a base portion of a methylated nucleotide
with an agent capable of cleaving the base portion of the
methylated nucleotide, whereby an abasic site is generated. In
another embodiment, the invention provides a method for labeling a
polynucleotide fragment, said method comprising labeling at an
abasic site of a polynucleotide fragment comprising the abasic
site, whereby a labeled polynucleotide fragment is generated,
wherein the abasic site is generated by cleaving a base portion of
a methylated nucleotide with an agent capable of cleaving the base
portion of the methylated nucleotide, and wherein the
polynucleotide fragment is generated by cleaving the phosphodiester
backbone of the polynucleotide comprising the abasic site at the
abasic site.
[0018] In one aspect, the invention provides methods for labeling
and fragmenting a polynucleotide comprising a methylated
nucleotide, said methods comprising: (a) contacting a
polynucleotide comprising (in some embodiments, suspected of
comprising) a methylated nucleotide with an agent (such as an
enzyme) capable of cleaving a base portion of an unmethylated
nucleotide (i.e., cleaving a base portion of the unmethylated
nucleotide), whereby an abasic site is generated, wherein the agent
(such as an enzyme) is not capable of cleaving a methylated
nucleotide; (b) cleaving the backbone of the polynucleotide
comprising the abasic site at the abasic site; and (c) contacting
the polynucleotide comprising the abasic site with an agent capable
of labeling the abasic site (i.e., labeling the abasic site),
whereby labeled polynucleotide fragments are generated. In some
embodiments, the agent capable of cleaving a base portion of an
unmethylated nucleotide comprises an enzyme. In one embodiment, the
unmethylated nucleotide is cytosine and the enzyme is cytosine
deaminase in conjunction with uracil DNA glycosylase. In some
embodiments, cleavage of a phosphodiester backbone of a
polynucleotide is performed with an agent selected from the group
consisting of an enzyme, a chemical agent, acidic conditions, basic
conditions, and heat. Two or more of the steps described above may
be performed simultaneously, or the steps may be performed
sequentially. For example, steps (a), (b), and (c) may be performed
simultaneously, steps (a) and (b) may be performed simultaneously,
or steps (b) and (c) may be performed simultaneously. When the
steps are performed sequentially, step (b) may be performed before
step (c) or step (c) may be performed before step (b).
[0019] In another aspect, the invention provides a method for
producing a labeled polynucleotide comprising a methylated
nucleotide, comprising: (a) cleaving a base portion of an
unmethylated nucleotide with an agent capable of cleaving the base
portion of the unmethylated nucleotide, whereby an abasic site is
generated, wherein the agent is not capable of cleaving the base
portion of a methylated nucleotide; and (b) labeling at the abasic
site, whereby a labeled polynucleotide is generated.
[0020] In another aspect, the invention provides a method for
fragmenting a polynucleotide comprising a methylated nucleotide,
comprising: (a) cleaving a base portion of an unmethylated
nucleotide with an agent capable of cleaving the base portion of
the unmethylated nucleotide, whereby an abasic site is generated,
wherein the agent is not capable of cleaving the base portion of a
methylated nucleotide; and (b) cleaving the backbone of the
polynucleotide comprising the abasic site, whereby polynucleotide
fragments are generated.
[0021] In another aspect, the invention provides method for
fragmenting and labeling a polynucleotide comprising a methylated
nucleotide, comprising: (a) incubating a reaction mixture, said
reaction mixture comprising: (i) a polynucleotide comprising a
methylated nucleotide; and (ii) an agent capable of cleaving a base
portion of a unmethylated nucleotide; wherein the incubation is
under conditions that permit cleavage of the base portion of the
unmethylated nucleotide, wherein the agent is not capable of
cleaving a methylated nucleotide, whereby a polynucleotide
comprising an abasic site is generated; (b) incubating a reaction
mixture, said reaction mixture comprising: (i) a polynucleotide
comprising an abasic site; and (ii) an agent capable of effecting
(generally, specific) cleavage of a phosphodiester backbone at the
abasic site; wherein the incubation is under conditions that permit
cleavage of the phosphodiester backbone at the abasic site; whereby
fragments of the polynucleotide are generated; (c) incubating a
reaction mixture, said reaction mixture comprising: (i) a
polynucleotide comprising an abasic site; and (ii) an agent capable
of labeling the abasic site; wherein the incubation is under
conditions that permit labeling at the abasic site; whereby labeled
fragments are generated.
[0022] In one embodiment, the invention provides a method for
fragmenting a polynucleotide comprising a methylated nucleotide,
said method comprising cleaving the phosphodiester backbone of a
polynucleotide comprising an abasic site at the abasic site,
wherein the abasic site is generated by cleaving a base portion of
an unmethylated nucleotide with an agent capable of cleaving the
base portion of the unmethylated nucleotide, whereby an abasic site
is generated, wherein the agent is not capable of cleaving a
methylated nucleotide. In another embodiment, the invention
provides a method for fragmenting and labeling a polynucleotide
comprising a methylated nucleotide, said method comprising cleaving
the phosphodiester backbone of a polynucleotide comprising an
abasic site at the abasic site, wherein the abasic site is
generated by cleaving a base portion of an unmethylated nucleotide
with an agent capable of cleaving the base portion of the
unmethylated nucleotide, whereby an abasic site is generated,
wherein the agent is not capable of cleaving a methylated
nucleotide, and labeling at the abasic site, whereby a labeled
polynucleotide fragment is generated. In another embodiment, the
invention provides a method for labeling a polynucleotide
comprising a methylated nucleotide, said method comprising labeling
at the abasic site of a polynucleotide comprising an abasic site,
whereby a labeled polynucleotide is generated, wherein the abasic
site is generated by cleaving a base portion of an unmethylated
nucleotide with an agent capable of cleaving the base portion of
the unmethylated nucleotide, whereby an abasic site is generated,
wherein the agent is not capable of cleaving a methylated
nucleotide. In another embodiment, the invention provides a method
for labeling a polynucleotide fragment, said method comprising
labeling at an abasic site of a polynucleotide fragment comprising
the abasic site, whereby a labeled polynucleotide fragment is
generated, wherein the abasic site is generated by cleaving a base
portion of an unmethylated nucleotide with an agent capable of
cleaving the base portion of the unmethylated nucleotide, and
wherein the polynucleotide fragment is generated by cleaving the
phosphodiester backbone of the polynucleotide comprising the abasic
site at the abasic site.
[0023] In another aspect, the invention provides methods using the
labeled and/or fragmented methylated polynucleotides generated
using the methods for labeling and/or fragmenting a methylated
polynucleotide described herein for, e.g., detection of methylation
(including presence and/or absence and/or quantity or level of
methylation), identification of methylated polynucleotide
sequences, isolation of methylated polynucleotide sequences,
characterization of methylated polynucleotide sequences, and other
applications as described herein. In one embodiment, the invention
provides a method for characterizing a methylated polynucleotide,
comprising detecting a polynucleotide fragment or a labeled
polynucleotide fragment produced by any of the methods described
herein, wherein detection of the polynucleotide fragment correlates
with presence, absence, sequence, or amount of the methylated
polynucleotide.
[0024] In another aspect, the invention provides methods for
labeling and fragmenting a polynucleotide comprising a canonical
nucleotide, said methods comprising: (a) cleavage of a base portion
of a canonical nucleotide present in a polynucleotide comprising
the canonical nucleotide with an agent (such as an enzyme) capable
of cleaving a base portion of the canonical nucleotide (whereby an
abasic site is generated); (b) cleavage of the phosphodiester
backbone at the abasic site, and (c) labeling at the abasic site,
whereby labeled polynucleotide fragments are generated. In some
embodiments, the agent capable of cleaving a base portion of a
canonical nucleotide comprises an enzyme. In one embodiment, the
canonical nucleotide is cytosine and the enzyme comprises cytosine
deaminase in conjunction with uracil DNA glycosylase. In some
embodiment, cleavage of the phosphodiester backbone of a
polynucleotide is performed with an agent selected from the group
consisting of an enzyme, a chemical agent, acidic conditions, basic
conditions, and heat. Two or more of the steps described above may
be performed simultaneously, or the steps may be performed
sequentially. For example, steps (a), (b), and (c) may be performed
simultaneously, steps (a) and (b) may be performed simultaneously,
or steps (b) and (c) may be performed simultaneously. When the
steps are performed sequentially, step (b) may be performed before
step (c) or step (c) may be performed before step (b).
[0025] In another aspect, the invention provides methods for
fragmenting and labeling a polynucleotide comprising a canonical
nucleotide: (a) incubating a reaction mixture, said reaction
mixture comprising: (i) a polynucleotide comprising a canonical
nucleotide; and (ii) an agent capable of specifically cleaving a
base portion of a canonical nucleotide; wherein the incubation is
under conditions that permit cleavage of the base portion of the
canonical nucleotide, whereby a polynucleotide comprising an abasic
site is generated; (b) incubating a reaction mixture, said reaction
mixture comprising: (i) a polynucleotide comprising an abasic site;
and (ii) an agent capable of effecting (generally, specific)
cleavage of a phosphodiester backbone at the abasic site; wherein
the incubation is under conditions that permit cleavage of the
phosphodiester backbone at the abasic site; whereby fragments of
the polynucleotide are generated; (c) incubating a reaction
mixture, said reaction mixture comprising: (i) a polynucleotide
comprising an abasic site; and (ii) an agent capable of labeling
the abasic site; wherein the incubation is under conditions that
permit labeling at the abasic site; whereby labeled fragments are
generated.
[0026] In another aspect, the invention provides methods for
labeling a polynucleotide comprising a canonical nucleotide, said
methods comprising (a) cleavage of a base portion of a canonical
nucleotide present in a polynucleotide comprising the canonical
nucleotide with an agent (such as an enzyme) capable of cleaving a
base portion of the canonical nucleotide (whereby an abasic site is
generated); and (b) labeling at the site of incorporation of the
canonical nucleotide (i.e., at the abasic site), whereby a labeled
polynucleotide(s) is generated.
[0027] In another aspect, the invention provides methods for
fragmenting a polynucleotide comprising a canonical nucleotide,
said methods comprising: (a) cleavage of a base portion of a
canonical nucleotide present in a polynucleotide comprising the
canonical nucleotide with an agent (such as an enzyme) capable of
cleaving a base portion of the canonical nucleotide (whereby an
abasic site is generated); and (b) cleavage of the phosphodiester
backbone at the abasic site, whereby polynucleotide fragments are
generated.
[0028] In one embodiment, the invention provides a method for
fragmenting a polynucleotide comprising a canonical nucleotide,
said method comprising cleaving the phosphodiester backbone of a
polynucleotide comprising an abasic site at the abasic site,
wherein the abasic site is generated by cleaving a base portion of
a canonical nucleotide with an agent capable of cleaving the base
portion of the canonical nucleotide, whereby an abasic site is
generated. In another embodiment, the invention provides a method
for fragmenting and labeling a polynucleotide comprising a
canonical nucleotide, said method comprising cleaving the
phosphodiester backbone of a polynucleotide comprising an abasic
site at the abasic site, wherein the abasic site is generated by
cleaving a base portion of a canonical nucleotide with an agent
capable of cleaving the base portion of the canonical nucleotide,
whereby an abasic site is generated, and labeling at the abasic
site, whereby a labeled polynucleotide fragment is generated. In
another embodiment, the invention provides a method for labeling a
polynucleotide comprising a canonical nucleotide, said method
comprising labeling at the abasic site of a polynucleotide
comprising an abasic site, whereby a labeled polynucleotide is
generated, wherein the abasic site is generated by cleaving a base
portion of a canonical nucleotide with an agent capable of cleaving
the base portion of the canonical nucleotide, whereby an abasic
site is generated. In another embodiment, the invention provides a
method for labeling a polynucleotide fragment, said method
comprising labeling at an abasic site of a polynucleotide fragment
comprising the abasic site, whereby a labeled polynucleotide
fragment is generated, wherein the abasic site is generated by
cleaving a base portion of a canonical nucleotide with an agent
capable of cleaving the base portion of a canonical nucleotide, and
wherein the polynucleotide fragment is generated by cleaving the
phosphodiester backbone of the polynucleotide comprising the abasic
site at the abasic site.
[0029] In some embodiments, the polynucleotide comprising the
canonical nucleotide (that is to be cleaved by the agent that
cleaves a base portion of the canonical nucleotide) can be single
stranded, double-stranded or partially double stranded. In some
embodiments, the polynucleotide comprises a cDNA. In other
embodiments, the polynucleotide comprises RNA, mRNA, genomic DNA,
or synthetic DNA. In other embodiments, the polynucleotide
comprises a cDNA library, a subtractive hybridization library, or a
genomic library.
[0030] In some embodiments, the invention provides methods wherein
the cleavage of the base portion of the cleavable canonical
nucleotide is under the same conditions (using the same agent) as
the cleavage of the backbone at the abasic site, and labeling at
the abasic site. In some embodiments, the reaction conditions are
acidic reaction conditions (such as pH 3, pH 3.5 or pH 4). In still
other embodiments, labeling and fragmentation (including cleavage
of the base portion of the canonical nucleotide and cleavage of the
phosphodiester backbone at an abasic site) are under the same
conditions (using the same agent). In still other embodiments, the
reaction conditions are acidic reaction conditions (such as pH 3,
pH 3.5 or pH 4).
[0031] The methods of the invention involving cleavage of a
cleavable canonical nucleotide include methods using the labeled
polynucleotide fragments and labeled polynucleotides produced by
the methods of the invention (so-called "applications"). The
invention provides methods to characterize (for example, detect
presence or absence of and/or quantify) a sequence of interest by
analyzing the labeled and/or fragmented products by
detection/quantification methods such as those based on array
technologies or solution phase technologies. In some embodiments,
the invention provides methods of detecting the presence or absence
of mutations.
[0032] In other embodiments, the invention provides methods of
producing a hybridization probe or target, hybridization using the
hybridization probes or targets; detection using the hybridization
probes or targets; characterizing and/or quantitating nucleic acid,
preparing a subtractive hybridization probe, comparative genomic
hybridization, and determining a gene expression profile, using the
labeled and/or fragmented nucleic acids generated by the methods of
the invention. Any of the methods described herein may be used to
generated labeled polynucleotides and/or polynucleotide fragments
which may be used as hybridization probes or targets. Labeled or
unlabeled targets produced by a method of the invention may be
hybridized to a probe.
[0033] The invention also provides methods for the generation of
polynucleotides, or fragments thereof, immobilized to a substrate
(surface), e.g., hybridization probes. In some embodiments, the
immobilized polynucleotide, or immobilized polynucleotide fragment
(in embodiments involving fragmentation) is labeled according to
the labeling methods described herein. These methods are suitable
for, for example, the production of microarrays or tagged
analytes.
[0034] As is evident to one skilled in the art, aspects that refer
to combining and incubating the resultant mixture also encompasses
method embodiments which comprise incubating the various mixtures
(in various combinations and/or subcombinations) so that the
desired products are formed. The reaction mixtures may be combined
(thus reducing the number of incubations) in any way, with one or
more reaction mixtures above combined. It is understood that any
combination of these incubation steps, and any single incubation
step, to the extent that the incubation is performed as part of any
of the methods described herein, fall within the scope of the
invention.
[0035] One or more steps may be combined and/or performed
sequentially (often in any order, as long as the requisite
product(s) are able to be formed), and, as is evident, the
invention includes various combinations of the steps described
herein. It is also evident, and is described herein, that the
invention encompasses methods in which the initial, or first, step
is any of the steps described herein. Methods of the invention
encompass embodiments in which later, "downstream" steps are an
initial step.
[0036] The invention also provides compositions, kits, complexes,
reaction mixtures and systems comprising various components (and
various combinations of the components) used in the methods
described herein. In one aspect, the invention provides a
composition comprising an agent capable of cleaving a base portion
of a methylated nucleotide to produce an abasic site on a
polynucleotide and an agent capable of labeling at an abasic site
on a polynucleotide. In one embodiment, the composition further
comprises an agent capable of cleaving a phosphodiester backbone of
a polynucleotide at an abasic site. In another aspect, the
invention provides a composition comprising an agent capable of
cleaving a base portion of a canonical nucleotide to produce an
abasic site on a polynucleotide and an agent capable of labeling at
an abasic site on a polynucleotide. In one embodiment, the
composition further comprises an agent capable of cleaving a
phosphodiester backbone of a polynucleotide at an abasic site on a
polynucleotide. In another aspect, the invention provides a
composition comprising a population of labeled and/or fragmented
polynucleotides produced by any of the methods described herein.
Compositions of the invention may also optionally further comprise
a composite primer comprising a DNA portion and a 5' RNA
portion.
[0037] In another aspect, the invention provides a kit comprising
an agent capable of cleaving a base portion of a methylated
nucleotide to produce an abasic site on a polynucleotide and an
agent capable of labeling at an abasic site on a polynucleotide. In
one embodiment, the kit comprises an agent capable of cleaving a
phosphodiester backbone of a polynucleotide at an abasic site. In
another aspect, the invention provides a kit comprising an agent
capable of cleaving a base portion of a canonical nucleotide to
produce an abasic site on a polynucleotide and an agent capable of
labeling at an abasic site on a polynucleotide. In one embodiment,
the kit comprises an agent capable of cleaving a phosphodiester
backbone of a polynucleotide at an abasic site. Kits of the
invention may also optionally further comprise a composite primer
comprising a DNA portion and a 5' RNA portion.
DESCRIPTION OF THE FIGURE
[0038] FIG. 1: is a photograph of a gel showing the labeled and
fragmented polynucleotide generated by acid-catalyzed cleavage
fragmentation and labeling of cDNA.
MODES FOR CARRYING OUT THE INVENTION
[0039] Methods of the Invention
[0040] I. Methods for Methylation Analysis
[0041] The invention provides methods and kits for analyzing DNA
methylation (including detecting and/or identifying methylated DNA
sequences). In one aspect, the methods comprise use of an agent
(such as an enzyme) that cleaves a base portion from a methylated
nucleotide (such as 5-methylcytosine), whereby an abasic site is
generated; cleavage of the phosphodiester backbone of the
polynucleotide comprising the abasic site at the abasic site; and
labeling at the abasic site, whereby labeled polynucleotide
fragments are generated. In another aspect, the methods comprise
use of an enzyme (such as cytosine deaminase in conjunction with
uracil N glycosylase (UNG)) that cleaves a base portion from an
unmethylated nucleotide, whereby an abasic site is generated,
wherein the enzyme is not capable of cleaving a methylated
nucleotide; cleavage of the phosphodiester backbone of the
polynucleotide comprising the abasic site at the abasic site; and
labeling at the abasic site, whereby labeled polynucleotide
fragments are generated.
[0042] Generally, the polynucleotide comprising a methylated
nucleotide (in some embodiments, suspected of comprising a
methylated nucleotide) is fragmented and labeled at the abasic site
(which is generated by cleavage of a base portion of the methylated
nucleotide). Thus, the methods of the invention are useful for,
e.g., detecting methylation (including presence and/or absence
and/or quantity or level of methylation), identifying methylated
polynucleotide sequences, isolating methylated polynucleotide
sequences, identifying and/or isolating methylatable polynucleotide
sequences, and other applications as described herein. The methods
of the invention generate labeled polynucleotide fragments which
are useful for, e.g., hybridization to a microarray and other uses
described herein.
[0043] In one aspect, the methods involve the following steps: (a)
contacting a polynucleotide comprising (in some embodiments,
suspected of comprising) a methylated nucleotide with an agent
capable of cleaving a base portion of the methylated nucleotide
(i.e., cleaving a base portion of the methylated nucleotide),
whereby an abasic site is generated; (b) cleaving the backbone of
the polynucleotide comprising the abasic site at the abasic site;
and (c) contacting the polynucleotide comprising the abasic site
with an agent capable of labeling the abasic site (i.e., labeling
the abasic site), whereby labeled polynucleotide fragments are
generated.
[0044] In another aspect, the methods involve the following steps:
(a) contacting a polynucleotide comprising (in some embodiments,
suspected of comprising) a methylated nucleotide with an agent
capable of cleaving a base portion of an unmethylated nucleotide
(i.e., cleaving a base portion of the unmethylated nucleotide),
whereby an abasic site is generated, wherein the agent is not
capable of cleaving a methylated nucleotide; (b) cleaving the
backbone of the polynucleotide comprising the abasic site at the
abasic site; and (c) contacting the polynucleotide comprising the
abasic site with an agent capable of labeling the abasic site
(i.e., labeling the abasic site), whereby labeled polynucleotide
fragments are generated.
[0045] In another aspect, the methods comprise the following steps:
(a) contacting a polynucleotide comprising (in some embodiments,
suspected of comprising) a methylated nucleotide with an enzyme
capable of cleaving a base portion of an unmethylated nucleotide
(i.e., cleaving a base portion of the unmethylated nucleotide),
whereby an abasic site is generated, wherein the enzyme is not
capable of cleaving a methylated nucleotide; (b) cleaving the
backbone of the polynucleotide comprising the abasic site at the
abasic site; and (c) contacting the polynucleotide comprising the
abasic site with an agent capable of labeling the abasic site
(i.e., labeling the abasic site), whereby labeled polynucleotide
fragments are generated. In some embodiments, the enzyme is
cytosine deaminase in conjunction with uracil DNA glycosylase.
[0046] In another aspect, the invention provides methods using the
labeled and/or fragmented methylated polynucleotides, for, e.g.,
detection of methylation (including presence and/or absence and/or
quantity or level of methylation), identification of methylated
polynucleotide sequences, isolation of methylated polynucleotide
sequences, characterization of methylated polynucleotide sequences,
and other applications as described herein.
[0047] II. Methods for Labeling and Fragmenting a Polynucleotide
Comprising a Cleavable Canonical Nucleotide, and Methods for
Labeling a Polynucleotide Comprising a Cleavable Canonical
Nucleotide
[0048] The invention provides novel methods and kits for labeling
and fragmenting a polynucleotide, and novel methods and kits for
labeling a polynucleotide. These methods are suitable for, for
example, generation of labeled polynucleotides, or labeled
polynucleotide fragments, for use as hybridization targets.
Generally, the polynucleotide is labeled at an abasic site present
in the polynucleotide, and fragmented at an abasic site present in
the polynucleotide (in embodiments involving fragmentation). The
abasic site present in the polynucleotide is generally prepared by
cleavage of a base portion of a cleavable canonical
(interchangeably termed "canonical") nucleotide present in the
polynucleotide.
[0049] Thus, in one aspect, the invention provides methods for
labeling and fragmenting a polynucleotide. The methods generally
comprise cleavage of a base portion of a canonical nucleotide
present in a polynucleotide comprising the canonical nucleotide
with an agent (such as an enzyme) capable of cleaving a base
portion of the canonical nucleotide (whereby an abasic site is
generated); cleavage of the phosphodiester backbone at the abasic
site, and labeling at the abasic site, whereby labeled
polynucleotide fragments are generated. In another aspect, the
invention provides methods for labeling a polynucleotide. The
methods generally comprise cleavage of a base portion of a
canonical nucleotide present in a polynucleotide comprising the
canonical nucleotide with an agent (such as an enzyme) capable of
cleaving a base portion of the canonical nucleotide (whereby an
abasic site is generated); and labeling at the site of
incorporation of the canonical nucleotide (i.e., at the abasic
site), whereby a labeled polynucleotide(s) is generated.
[0050] The polynucleotide comprising the canonical nucleotide (that
is to be cleaved by the agent that cleaves a base portion of the
canonical nucleotide) can be single stranded, double-stranded or
partially double stranded. In some embodiments, the polynucleotide
comprises a cDNA. In other embodiments, the polynucleotide
comprises RNA, mRNA, genomic DNA, or synthetic DNA. In other
embodiments, the polynucleotide comprises a cDNA library, a
subtractive hybridization library, or a genomic library.
[0051] It is understood that a polynucleotide comprising a
canonical nucleotide can be a multiplicity (from small to very
large) of different polynucleotide molecules. Such populations can
be related in sequence (e.g., members of a gene family or
superfamily) or extremely diverse in sequence (e.g., generated from
all mRNA, generated from all genomic DNA, etc.). Polynucleotides
can also correspond to single sequences (which can be part or all
of a known gene, including, e.g., a coding region, genomic region,
gene locus, etc.).
[0052] A base portion of the canonical nucleotide is cleaved by an
agent (such as an enzyme) capable of cleaving a base portion of a
canonical nucleotide. Such agents are known in the art and
described herein. In some embodiments, the agent capable of
cleaving a base portion of a canonical nucleotide is cytosine
deaminase (generally in conjunction with UNG), acidic conditions or
treatment with an alkylating agent. In some embodiments, the agent
is an enzyme. In other embodiments, the agent is a chemical agent.
In still other embodiments, the agent is reaction conditions (e.g.,
acidic conditions).
[0053] In some embodiments, the invention provides methods wherein
the cleavage of the base portion of the cleavable canonical
nucleotide is under the same conditions (using the same agent) as
the cleavage of the backbone at the abasic site, and labeling at
the abasic site. In some embodiments, the reaction conditions are
acidic reaction conditions (such as pH 3, pH 3.5 or pH 4). In still
other embodiments, labeling and fragmentation (including cleavage
of the base portion of the canonical nucleotide and cleavage of the
phosphodiester backbone at an abasic site) are under the same
conditions (using the same agent). In still other embodiments, the
reaction conditions are acidic reaction conditions (such as pH 3,
pH 3.5 or pH 4).
[0054] The methods of the invention involving cleavage of a
cleavable canonical nucleotide include methods using the labeled
polynucleotide fragments and labeled polynucleotides produced by
the methods of the invention (so-called "applications"). The
invention provides methods to characterize (for example, detect
presence or absence of and/or quantify) a sequence of interest by
analyzing the labeled and/or fragmented products by
detection/quantification methods such as those based on array
technologies or solution phase technologies. In some embodiments,
the invention provides methods of detecting the presence or absence
of mutations. The invention provides methods of detection whrein
detection correlates with presence, absence, sequence, and/or
amount of polynucleotide. These detection methods apply to all
methods described herein (based on cleavage of methylated
nucleotides, unmethylated nucleotides, as well as canonical
nucleotides).
[0055] In other embodiments, the invention provides methods of
producing a hybridization target, hybridization to a hybridization
probe; detection of the target hybridized to the probe;
characterizing and/or quantitating nucleic acid, preparing a
subtractive hybridization probe, comparative genomic hybridization,
and determining a gene expression profile, using the labeled and/or
fragmented nucleic acids generated by the methods of the
invention.
[0056] The invention also provides methods for the generation of
polynucleotides, or fragments thereof, immobilized to a substrate
(surface). In some embodiments, the immobilized polynucleotide, or
immobilized polynucleotide fragment (in embodiments involving
fragmentation) is labeled according to the labeling methods
described herein. These methods are suitable for, for example, the
production of microarrays or tagged analytes.
[0057] The methods of the invention include methods using the
immobilized polynucleotides, or immobilized polynucleotide
fragments produced by the methods of the invention (so-called
"applications"). In some embodiments, the invention provides
methods of detecting nucleic acid sequence mutations.
[0058] The invention also provides methods to characterize (for
example, detect presence or absence of and/or quantify) a sequence
of interest using the immobilized polynucleotides, or fragments
thereof.
[0059] In another embodiment, the invention provides methods of
determining a gene expression profile, using the immobilized
polynucleotides, or fragments thereof, generated by the methods of
the invention.
[0060] General Techniques
[0061] The practice of the invention will employ, unless otherwise
indicated, conventional techniques of molecular biology (including
recombinant techniques), microbiology, cell biology, biochemistry,
and immunology, which are within the skill of the art. Such
techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook
et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984);
"Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in
Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular
Biology" (F. M. Ausubel et al., eds., 1987, and periodic updates);
"PCR: The Polymerase Chain Reaction", (Mullis et al., eds.,
1994).
[0062] Primers, oligonucleotides and polynucleotides employed in
the invention can be generated using standard techniques known in
the art.
[0063] Definitions
[0064] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA. The nucleotides can be deoxyribonucleotides, modified
nucleotides or bases, and/or their analogs, or any substrate that
can be incorporated into a polymer by DNA polymerase. In
embodiments involving methylation analysis, nucleotides include
methylated nucleotides. A polynucleotide may comprise modified
(altered) nucleotides, such as, for example, modification to the
nucleotide structure and or modification to the phosphodiester
backbone. As discussed herein modified nucleotide can be a
canonical (cleavable) nucleotides, though, generally, a canonical
nucleotide according to the methods for cleavage of a canonical
nucleotide includes an unmodified base portion (i.e., unmodified
adenine, cytosine, guanine and thymine base). It is understood,
however, that modified nucleotides that are not (canonical)
cleavable nucleotide under the reaction conditions used in the
methods of the invention, if present, generally should not affect
the ability of the polynucleotide to undergo cleavage of a base
portion of the cleavable canonical nucleotide, such that an abasic
site is generated, and/or cleavage of a phosphodiester backbone at
an abasic site, such that fragments are generated, and/or
immobilization of a polynucleotide (or fragment thereof) to a
substrate, as described herein. If present, modification to the
nucleotide structure may be imparted before or after assembly of
the polymer. The sequence of nucleotides may be interrupted by
non-nucleotide components. A polynucleotide may be further modified
after polymerization, such as by conjugation with a labeling
component. Other types of modifications include, for example,
"caps", substitution of one or more of the naturally occurring
nucleotides with an analog, internucleotide modifications such as,
for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, poly-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). It is understood that internucleotide
modifications may, e.g., alter the efficiency and/or kinetics of
cleavage of the phosphodiester backbone (as when, for example a
phosphodiester backbone is cleaved at an abasic site, as described
herein). Further, any of the hydroxyl groups ordinarily present in
the sugars may be replaced, for example, by phosphonate groups,
phosphate groups, protected by standard protecting groups, or
activated to prepare additional linkages to additional nucleotides.
The 5' and 3' terminal OH can be phosphorylated or substituted with
amines or organic capping group moieties of from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides can also contain analogous forms
of ribose or deoxyribose sugars that are generally known in the
art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs. One or more phosphodiester linkages
may be replaced by alternative linking groups. These alternative
linking groups include, but are not limited to, embodiments wherein
phosphate is replaced by P(O)S("thioate"), P(S)S ("dithioate"),
"(O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or CH.sub.2
("formacetal"), in which each R or R' is independently H or
substituted or unsubstituted alkyl (1-20 C) optionally containing
an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl
or araldyl. Not all linkages in a polynucleotide need be identical.
The preceding description applies to all polynucleotides referred
to herein, including DNA. It is understood, however, that modified
nucleotides and/or internucleotide linkages, if present, generally
should not affect the ability of the polynucleotide to undergo
cleavage of a base portion of a canonical nucleotide, such that an
abasic site is generated, and/or the ability of a polynucleotide to
undergo cleavage of a phosphodiester backbone at an abasic site,
such that fragments are generated, and/or the ability of a
polynucleotide to be immobilized at an abasic site (such as an
abasic site at an end of a polynucleotide and/or an abasic site
that is not at an end of a polynucleotide) to a surface, as
described herein.
[0065] "Oligonucleotide," as used herein, generally refers to
short, generally single stranded, generally synthetic
polynucleotides that are generally, but not necessarily, less than
about 200 nucleotides in length. The terms "oligonucleotide" and
"polynucleotide" are not mutually exclusive. The description above
for polynucleotides is equally and fully applicable to
oligonucleotides.
[0066] A "primer," as used herein, refers to a nucleotide sequence
(a polynucleotide), generally with a free 3'--OH group, that
hybridizes with a template sequence (such as a template RNA, or a
primer extension product) and is capable of promoting
polymerization of a polynucleotide complementary to the template. A
"primer" can be, for example, an oligonucleotide. It can also be,
for example, a sequence of the template (such as a primer extension
product or a fragment of an RNA template created following cleavage
of a template RNA in an RNA-DNA complex by RNase H or other agent)
that is hybridized to a sequence in the template itself (for
example, as a hairpin loop), and that is capable of promoting
nucleotide polymerization by DNA polymerase. Thus, a primer can be
an exogenous (e.g., added) primer or an endogenous (e.g., template
fragment) primer.
[0067] A "complex" is an assembly of components. A complex may or
may not be stable and may be directly or indirectly detected. For
example, as is described herein, given certain components of a
reaction, and the type of product(s) of the reaction, existence of
a complex can be inferred. For purposes of this invention, a
complex is generally an intermediate with respect to the final
polynucleotide fragments, labeled polynucleotide, labeled
polynucleotide fragments, and/or immobilized polynucleotide or
fragment thereof.
[0068] A "fragment" of a polynucleotide or oligonucleotide is a
contiguous sequence of 2 or more bases. In other embodiments, a
fragment (also termed "region" or "portion") is any of about 3,
about 5, about 10, about 15, about 20, about 25, about 30 about 35
about 40, about 50, about 65, about 75, about 85, about 100, about
125, about 150, about 175, about 200, about 225, about 250, about
300, about 350, about 400, about 450, about 500, about 550, about
600, about 650 or more nucleotides in length. In some embodiments,
the fragments can be at least about 3, about 5, about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 50, about
65, about 75, about 85, about 100, about 125, about 150, about 175,
about 200, about 225, about 250, about 300, about 350, about 400,
about 450, about 500, about 550, about 600, about 650 or more
nucleotides in length. In other embodiments, the fragments can be
less than about 3, about 5, about 10, about 15, about 20, about 25,
about 30 about 35 about 40, about 50, about 65, about 75, about 85,
about 100, about 125, about 150, about 175, about 200, about 225,
about 250, about 300, about 350, about 400, about 450, about 500,
about 550, about 600, about 650 or more nucleotides in length. In
some embodiments, these fragment lengths represent an average size
in the population of fragments generated using the methods of the
invention.
[0069] A "reaction mixture" is an assemblage of components, which,
under suitable conditions, react to form a complex (which may be an
intermediate) and/or a product(s).
[0070] "A", "an" and "the", and the like, unless otherwise
indicated include plural forms. "A" fragment means one or more
fragments. "A" methylated nucleotide means one or more methylated
nucleotides. "An" enzyme means one or more than one enzyme.
[0071] "Comprising" means including in accordance with
well-established principles of patent law (i.e., open
language).
[0072] Conditions that "allow" an event to occur or conditions that
are "suitable" for an event to occur, such as polynucleotide
synthesis, cleavage of a base portion of a canonical nucleotide,
cleavage of a base portion of a methylated nucleotide, cleavage of
a phosphodiester backbone at an abasic site, and the like, or
"suitable" conditions are conditions that do not prevent such
events from occurring. Thus, these conditions permit, enhance,
facilitate, and/or are conducive to the event. Such conditions,
known in the art and described herein, depend upon, for example,
the nature of the polynucleotide sequence, temperature, and buffer
conditions. These conditions also depend on what event is desired,
such as polynucleotide synthesis, cleavage of a base portion of a
canonical nucleotide, cleavage of a base portion of a methylated
nucleotide, cleavage of a phosphodiester backbone at an abasic
site, labeling an abasic site, immobilizing a polynucleotide
fragment or a polynucleotide, etc.
[0073] "Microarray" and "array," as used interchangeably herein,
comprise a surface with an array, preferably ordered array, of
putative binding (e.g., by hybridization) sites for a biochemical
sample (target) which often has undetermined characteristics.
[0074] The term "3'" generally refers to a region or position in a
polynucleotide or oligonucleotide 3' (downstream) from another
region or position in the same polynucleotide or oligonucleotide.
"3'" may also refer to the 3' end of a polynucleotide or
oligonucleotide.
[0075] The term "5'" generally refers to a region or position in a
polynucleotide or oligonucleotide 5' (upstream) from another region
or position in the same polynucleotide or oligonucleotide. "5'" may
also refer to the 5' end of a polynucleotide or
oligonucleotide.
[0076] The terms "3'-DNA portion," "3'-DNA region," "3'-RNA
portion," and "3'-RNA region," refer to the portion or region of a
polynucleotide or oligonucleotide located towards the 3' end of the
polynucleotide or oligonucleotide, and may or may not include the
3' most nucleotide(s) or moieties attached to the 3' most
nucleotide of the same polynucleotide or oligonucleotide. The 3'
most nucleotide(s) can be preferably from about 1 to about 50, more
preferably from about 10 to about 40, even more preferably from
about 20 to about 30 nucleotides. In some embodiments, a 3' portion
can be any of at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20,
30, or 40 nucleotides. In some embodiments, a 3' portion can be
about 1 to about 20 nucleotides, about 5 to about 20 nucleotides,
or about 5 to about 50 nucleotides.
[0077] The terms "5'-DNA portion," "5'-DNA region," "5'-RNA
portion," and "5'-RNA region," refer to the portion or region of a
polynucleotide or oligonucleotide located towards the 5' end of the
polynucleotide or oligonucleotide, and may or may not include the
5' most nucleotide(s) or moieties attached to the 5' most
nucleotide of the same polynucleotide or oligonucleotide. The 5'
most nucleotide(s) can be preferably from about 1 to about 50, more
preferably from about 10 to about 40, even more preferably from
about 20 to about 30 nucleotides. In some embodiments a 5' portion
can be at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20,
30, or 40 nucleotides. In some embodiments, a 5' portion can be
about 1 to about 20 nucleotides, about 5 to about 20 nucleotides,
or about 5 to about 50 nucleotides.
[0078] As used herein in the context of methods for labeling and/or
fragmenting a canonical nucleotide, "canonical" nucleotide means a
nucleotide comprising one of the four common nucleic acid bases
adenine, cytosine, guanine and thymine that are commonly found in
DNA. The term also encompasses the respective deoxyribonucleosides,
deoxyribonucleotides or 2'-deoxyribonucleoside-5'-triphosphates
that contain one of the four common nucleic acid bases adenine,
cytosine, guanine and thymine.
[0079] The term "analyte" as used herein refers to a substance to
be detected or assayed by the method of the present invention, for
example, a compound whose properties, location, quantity and/or
identity is desired to be characterized. Typical analytes may
include, but are not limited to proteins, peptides, nucleic acid
segments, cells, microorganisms and fragments and products thereof,
organic molecules, inorganic molecules, or any substance for which
immobilization sites for binding partner(s) can be developed. As
this disclosure clearly conveys, an analyte is a substrate.
[0080] As used herein, an "abasic site" refers to the site of a
canonical nucleotide following treatment with an agent capable of
effecting cleavage of a base portion of the canonical nucleotide
(in embodiments relating to labeling and/or fragmentation of a
polynucleotide comprising a canonical nucleotide), or the site of
the methylated nucleotide following treatment with an agent capable
of effecting cleavage of a base portion of the methylated
nucleotide (in embodiments relating to labeling and/or
fragmentation of a polynucleotide comprising a methylated
nucleotide) or the site of the unmethylated nucleotide following
treatment with an agent capable of effecting cleavage of a base
portion of the methylated nucleotide (in embodiments relating to
labeling and/or fragmentation of a polynucleotide comprising a
methylated nucleotide, said methods comprising cleavage of a base
portion of a unmethylated nucleotide. An abasic site
(interchangeably termed "AP site") can comprise a hemiacetal ring
and/or an aldehyde moiety, and lacks a base portion of the
canonical nucleotide. As used herein, "abasic site" encompasses, in
embodiments relating to labeling and/or fragmentation of a
polynucleotide comprising a canonical nucleotide, any chemical
structure remaining following treatment of a canonical nucleotide
(present in a polynucleotide chain) with an agent (e.g., an enzyme,
chemical reagent, heat, acidic conditions, and/or basic conditions)
capable of effecting cleavage of a base portion of a canonical
nucleotide, and, in embodiments relating to labeling and/or
fragmentation of a polynucleotide comprising a methylated
nucleotide, any chemical structure remaining following treatment of
a methylated nucleotide (present in a polynucleotide chain) (or, in
some embodiments, treatment of an unmethylated nucleotide) with an
agent (e.g., an enzyme, chemical reagent, heat, acidic conditions,
and/or basic conditions) capable of effecting cleavage of a base
portion of a methylated nucleotide (or, in some embodiments, an
unmethylated nucleotide). An abasic site may also occur in a
natural polynucleotide, indicating damage to the
polynucleotide.
[0081] As used herein, "labeling at an abasic site" means
association of a label with, in embodiments relating to labeling
and/or fragmentation of a polynucleotide comprising a canonical
nucleotide, any chemical structure remaining following removal of a
base portion (including the entire base) of a canonical nucleotide
(present in a polynucleotide chain) by treatment with an agent
(e.g., an enzyme, or heat)) capable of effecting cleavage of a base
portion of a canonical nucleotide, or, in embodiments relating to
labeling and/or fragmentation of a polynucleotide comprising a
methylated nucleotide, treatment with an agent capable of effecting
cleavage of a base portion of a methylated nucleotide (or, in some
embodiments, an unmethylated nucleotide). In one embodiment, a
reactive aldehyde form of a hemiacetal ring in an abasic site is
labeled. In other embodiments involving cleavage of a base portion
of a canonical nucleotide, the label associates with a chemical
structure remaining following treatment of a canonical nucleotide
(present in a polynucleotide chain) with an agent (e.g., an enzyme,
or heat or basic conditions) capable of effecting cleavage of a
base portion of a canonical nucleotide and treatment of
polynucleotide comprising an abasic site with an agent capable of
effecting cleavage of the backbone at the abasic site (as described
herein), or, in embodiments involving cleavage of a base portion of
a methylated nucleotide, the label associates with a chemical
structure remaining following treatment of a methylated nucleotide
(present in a polynucleotide chain) (in some embodiments, an
unmethylated nucleotide) with an agent (e.g., an enzyme, or heat or
basic conditions) capable of effecting cleavage of a base portion
of a methylated nucleotide (in some embodiments, an unmethylated
nucleotide) and treatment of polynucleotide comprising an abasic
site with an agent capable of effecting cleavage of the backbone at
the abasic site (as described herein).
[0082] As used herein, cleavage of a backbone (e.g. phosphodiester
backbone) "at" an abasic site means cleavage of the phosphodiester
linkage 3' to the abasic site or 5' to the abasic site, or both. As
the disclosure herein conveys, "at" an abasic site refers to
proximate or near location (such as immediately 3' or immediately
5'). In still other embodiments, more complex forms of cleavage are
possible, for example, cleavage such that cleavage of the
phosphodiester backbone and cleavage of (a portion of) the abasic
site results.
[0083] As used herein, a "label" (interchangeably called a
"detectable moiety") refers to a moiety that is associated or
covalently linked with a polynucleotide (interchangeably called
"labeling"). The labeled polynucleotide may be directly or
indirectly detected, generally through a detectable signal. The
detectable moiety (label) can be covalently attached (or
associated) either directly or indirectly through a non-interfering
linkage group with other moieties capable of specifically
associating with one or more sites to be labeled. The detectable
moiety (label) may be covalently linked or non-covalently
associated as well as directly or indirectly associated.
[0084] Methods for Labeling and/or Fragmenting a Polynucleotide
Comprising a Methylated Nucleotide
[0085] The following are examples of the methods of the invention.
It is understood that various other embodiments may be practiced,
given the general description provided herein. For example,
reference to using an agent capable of cleaving a base portion of
the methylated nucleotide means that any of the agents capable of
cleaving a base portion of the methylated nucleotide described
herein may be used.
[0086] A. Methods for Labeling and Fragmenting a Polynucleotide
Comprising a Methylated Nucleotide and Analyzing DNA
Methylation
[0087] The invention provides methods for analyzing DNA methylation
(including detecting and/or identifying methylated DNA sequences).
Generally, the methods comprise use of an agent (such as an enzyme)
that cleaves a base portion from a methylated nucleotide (such as
5-methylcytosine), whereby an abasic site is generated; cleavage of
the phosphodiester backbone of the polynucleotide comprising the
abasic site at the abasic site; and labeling at the abasic site,
whereby labeled polynucleotide fragments are generated. Generally,
the polynucleotide comprising a methylated nucleotide (in some
embodiments, suspected of comprising a methylated nucleotide) is
fragmented and labeled at the abasic site (which is generated by
cleavage of a base portion of the methylated nucleotide). Thus, the
methods of the invention are useful for, e.g., detecting
methylation (including presence and/or absence and/or quantity or
level of methylation), identifying methylated polynucleotide
sequences, isolating methylated polynucleotide sequences,
identifying and/or isolating methylatable polynucleotide sequences,
and other applications as described herein. The methods of the
invention generate labeled polynucleotide fragments which are
useful for, e.g., hybridization to a microarray and other uses
described herein.
[0088] In one aspect, the methods involve the following steps: (a)
contacting a polynucleotide comprising (in some embodiments,
suspected of comprising) a methylated nucleotide with an agent
capable of cleaving a base portion of the methylated nucleotide
(i.e., cleaving a base portion of the methylated nucleotide),
whereby an abasic site is generated; (b) cleaving the backbone of
the polynucleotide comprising the abasic site at the abasic site;
and (c) contacting the polynucleotide comprising the abasic site
with an agent capable of labeling the abasic site (i.e., labeling
the abasic site), whereby labeled polynucleotide fragments are
generated.
[0089] Generally, native methylation status is analyzed (i.e.,
methylation status or pattern as exists in vivo, or substantially
similar to the in vivo status or pattern), wherein the
polynucleotide is directly harvested and/or isolated from a
biological sample (such as a tissue or cell culture), though other
embodiments are contemplated (including embodiments comprising
detection and/or identification of methylatable sequences) in which
the polynucleotide is from any source (i.e., genomic DNA, or a
source other than genomic DNA) and the polynucleotide is methylated
prior to labeling and fragmenting as described above). Thus,
methylation status may be the result of in vivo processes, or of
experimental manipulations (e.g., deliberate exposure to a putative
DNA damaging agent, or a putative demethylating agent). The methods
of the invention (i.e., the methods comprising cleavage of a base
portion of a methylated nucleotide; cleavage of the backbone at the
abasic site and/or labeling at the abasic site) specifically
exclude methods comprising synthesis of a polynucleotide comprising
a non-canonical nucleotide as disclosed in co-pending co-owned U.S.
patent application Ser. No. 10/441,663 (publication no.
2004/0005614). Thus, the polynucleotide comprising a methylated
nucleotide (or suspected of comprising a methylated nucleotide), as
that term is used herein, does not encompass (specifically
excludes) a polynucleotide comprising a non-canonical nucleotide,
wherein the polynucleotide was generated by a method comprising
synthesizing a polynucleotide comprising a non-canonical nucleotide
from a template (i.e., such that the non-canonical nucleotide is
incorporated during template-dependent synthesis of the
polynucleotide comprising a non-canonical nucleotide.)
[0090] The polynucleotide to be analyzed for methylation status
(e.g., the polynucleotide suspected of comprising a methylated
nucleotide and/or the polynucleotide comprising a methylated
nucleotide) may be any polynucleotide from which labeled
polynucleotide fragments are desired to be produced, including
double-stranded, partially double-stranded, and single-stranded
nucleic acids from any source in purified or unpurified form, which
can be DNA (dsDNA and ssDNA) or RNA, including tRNA, mRNA, rRNA,
mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RNA
hybrids, or mixtures thereof, genes, chromosomes, the genomes of
biological material such as plants, animals, humans, and fragments
thereof (including fragments comprising hypermethylated CpG
islands, centromeric regions, and other hypermethylated regions),
though other embodiments are contemplated, as described herein. The
polynucleotide can be known or unknown and may contain more than
one sequence of interest, each of which may be the same or
different from each other. The polynucleotide can be a
sub-population of nucleic acids, for example, a subtractive
hybridization probe, total genomic DNA, or restriction
fragments.
[0091] Obtaining and purifying nucleic acids use standard
techniques in the art. Generally, the polynucleotide is DNA,
though, as noted herein, the polynucleotide can comprise altered
and/or modified nucleotides, internucleotide linkages,
ribonucleotides, etc. As generally used herein, it is understood
that "DNA" applies to polynucleotide embodiments. The
polynucleotide suspected of comprising a methylated nucleotide can
be processed or modified prior to analysis, for example, cleavage
using restriction enzymes or other means known in the art, such as
shearing, and using chemical or enzymatic methylation or
demethylation as known in the art.
[0092] For simplicity, the polynucleotide comprising (or suspected
of comprising) a methylated nucleotide is described as a single
nucleic acid. It is understood that the polynucleotide is generally
a population of polynucleotides (from a few to a multiplicity to a
very large multiplicity of polynucleotides). It is further
understood that a polynucleotide comprising a methylated nucleotide
can be a multiplicity (from small to very large) of different
polynucleotide molecules. Such populations can be related in
sequence (e.g., member of a gene family or superfamily) or
extremely diverse in sequence (such as all genomic DNA).
Polynucleotides can also correspond to single sequence (which can
be part or all of a known gene, for example a coding region,
genomic portion, etc.). In some embodiments, the polynucleotide
comprises a gene, a gene locus, a specific CpG dinucleotide, a CpG
island, and/or a centromeric region.
[0093] For simplicity, individual steps of the labeling and
fragmentation method are discussed herein. It is understood,
however, that the steps may be performed simultaneously and/or in
varied order, as discussed herein.
[0094] It is further understood that for convenience, methods
involving cleavage of a base portion of a methylated nucleotide,
and methods involving cleavage of a base portion of a
non-methylated nucleotide (wherein the agent that cleaves a base
portion of a nonmethylated nucleotide (interchangeably termed
"unmethylated" nucleotide) is generally not capable of cleaving the
base portion of a methylated nucleotide) (described below) are
described separately. It is understood that the methods may be
combined, performed separately, and/or performed sequentially.
[0095] 1. Cleaving a Base Portion of a Methylated Nucleotide to
Create an Abasic Site
[0096] In aspects involving cleavage of a base portion of a
methylated nucleotide, the polynucleotide comprising a methylated
nucleotide (in some embodiments, suspected of comprising a
methylated nucleotide) is treated with an agent, such as an enzyme,
capable of generally, specifically, or selectively cleaving a base
portion of the methylated deoxyribonucleoside to create an abasic
site. As used herein, "abasic site" encompasses any chemical
structure remaining following removal of a base portion (including
the entire base) of a methylated nucleotide with an agent capable
of cleaving a base portion of a methylated nucleotide, e.g., by
treatment of a methylated nucleotide (present in a polynucleotide
chain) with an agent (e.g., an enzyme) capable of effecting
cleavage of a base portion of a methylated nucleotide. In some
embodiments, the agent (such as an enzyme) catalyzes hydrolysis of
the bond between the base portion of the methylated nucleotide and
a sugar in the methylated nucleotide to generate an abasic site
comprising a hemiacetal ring and lacking the base (interchangeably
called "AP" site), though other cleavage products are contemplated
for use in the methods of the invention. Generally, the methods
involving cleavage of a base portion of a methylated nucleotide are
suitable for use with polynucleotides comprising a low frequency of
methylated nucleotides (i.e., generally, not hypermethylated
regions, such a CpG islands and the like), though other uses are
contemplated.
[0097] The polynucleotide comprising (in some embodiments,
suspected of comprising) a methylated nucleotide is treated with an
agent, such as an enzyme, capable of generally, specifically, or
selectively cleaving a base portion of the methylated
deoxyribonucleoside to create an abasic site. As used herein,
"abasic site" encompasses any chemical structure remaining
following removal of a base portion (including the entire base) of
a methylated nucleotide with an agent capable of cleaving a base
portion of a methylated nucleotide, e.g., by treatment of a
methylated nucleotide (present in a polynucleotide chain) with an
agent (e.g., an enzyme, acidic conditions, or a chemical reagent)
capable of effecting cleavage of a base portion of a methylated
nucleotide. In some embodiments, the agent (such as an enzyme)
catalyzes hydrolysis of the bond between the base portion of the
methylated nucleotide and a sugar in the canonical nucleotide to
generate an abasic site comprising a hemiacetal ring and lacking
the base (interchangeably called "AP" site), though other cleavage
products are contemplated for use in the methods of the
invention.
[0098] In some embodiments, the methylated nucleotide is
5-methylcytosine. In some embodiments, 5-methylcytosine is present
as a methylated CpG dinucleotide. The CpG dinucleotide may be fully
or hemi-methylated. In other embodiments, the methylated nucleotide
is 3-methyladenine. In other embodiments, the methylated nucleotide
is 7-methyladenine and/or 3-methylguanine.
[0099] Suitable agents and reaction conditions for cleavage of base
portions of methylated nucleotides are known in the art, and
include: 5-methylcytosine DNA glycosylase (5-MCDG), which cleaves
the base portion of 5-methylcytosine (5-MeC) from the DNA backbone
(Wolffe et al., Proc. Nat. Acad. Sci. USA 96:5894-5896, 1999);
3-methyladenosine-DNA glycosylase I, which cleaves the base portion
of 3-methyl adenosine from the DNA backbone (see, e.g. Hollis et al
(2000) Mutation Res. 460: 201-210); and/or 3-methyladenosine DNA
glycosylase II, which cleaves the base portion of
3-methyladenosine, 7-methylguanine, 7-methyladenosine,
and/3-methylguanine from the DNA backbone. See McCarthy et al
(1984) EMBO J. 3:545-550. Multifunctional and mono-functional forms
of 5-MCDG have been described. See Zhu et al., Proc. Natl. Acad.
Sci. USA 98:5031-6, 2001; Zhu et al., Nuc. Acid Res. 28:4157-4165,
2000; and Nedderrnann et al., J. B. C. 271:12767-74, 1996
(describing bifunctional 5-MCDG; Vairapandi & Duker, Oncogene
13:933-938, 1996; Vairapandi et al., J. Cell. Biochem. 79:249-260,
2000 (describing mono-functional enzyme comprising 5-MCDG
activity). In some embodiments, 5-MCDG preferentially cleaves fully
methylated polynucleotide sites (e.g., CpG dinucleotides), and in
other embodiments, 5-MCDG preferentially cleaves a hemi-methylated
polynucleotide. For example, mono-functional human 5-methylcytosine
DNA glycosylase cleaves DNA specifically at fully methylated CpG
sites, and is relatively inactive on hemimethylated DNA (Vairapandi
& Duker, supra; Vairapandi et al., supra). By contrast, chick
embryo 5-methylcytosine-DNA glycosylase has greater activity
directed to hemimethylated methylation sites. In some embodiments,
the activity of 5-MCDG is potentiated (increased or enhanced) with
accessory factors, such as recombinant CpG-rich RNA, ATP, RNA
helicase enzyme, and proliferating cell nuclear antigen (PCNA). See
U.S. Patent Publication No. 20020197639 A1. One or more agents may
be used. In some embodiments, the one or more agents cleave a base
portion of the same methylated nucleotide. In other embodiments,
the one or more agents cleave a base portion of different
methylated nucleotides. Treatment with two or more agents may be
sequential or simultaneous.
[0100] As is evident, in some embodiments, dUTP is generated as an
intermediate and cleavage of a base portion of dUTP is necessary to
generate the abasic site. Methods for cleaving a base portion of
dUTP are known in the art. See, e.g., Lindahl, PNAS (1974)
71(9):3649-3653; Jendrisak, U.S. Pat. No. 6,190,865 B1; U.S. Pat.
No. 5,035,996; U.S. Pat. No. 5,418,149; Sartori et al (2002) EMBO J
21:3182-3191. Thus, in some embodiments, an agent that cleaves a
base portion of a methylated nucleotides (such as an enzyme, such
as 5-MCDG) is used in conjunction with UNG to generate an abasic
site from the methylated nucleotide. As used herein, "in
conjunction" encompasses simultaneous treatment (e.g., when 5-MCDG
and UNG cleavage occurs in the same reaction mixture) and/or
treatment at different times (e.g., when 5-MCDG and UNG treatment
is conducted sequentially).
[0101] In some embodiments, the agent that cleaves the base portion
of the methylated nucleotide is the same agent that cleaves a
phosphodiester backbone at the abasic site.
[0102] In some embodiments, cleavage of the base portion of the
methylated nucleotides is general, specific or selective cleavage
(in the sense that the agent (such as an enzyme) capable of
cleaving a base portion of a methylated nucleotide generally,
specifically or selectively cleaves the base portion of a
particular methylated nucleotide), whereby about any of 98%, 95%,
90%, 85%, or 80% of the base portions cleaved are base portions of
methylated nucleotides. However, extent of cleavage can be less.
Thus, reference to specific cleavage is exemplary.
[0103] As noted herein, for convenience, cleavage of a base portion
of a methylated nucleotide (whereby an abasic site is generated)
has been described as a separate step. It is understood that this
step may be performed simultaneously with cleavage of the backbone
at an abasic site (fragmentation) and/or labeling at an abasic
site.
[0104] It is understood that the frequency (or spacing) of abasic
sites in the resulting polynucleotide comprising an abasic site
(following cleavage of a base portion of a methylated nucleotide,
and thus the average size of fragments generated using the methods
of the invention (i.e., following cleavage of a phosphodiester
backbone at an abasic site), is controlled by variables known in
the art, including: frequency of methylated nucleotide(s) in the
polynucleotide (or other measures of nucleotide content of a
sequence, such as average G-C content), length of the
polynucleotide comprising a methylated nucleotide, and the reaction
conditions used during generation of abasic site, as is further
discussed herein. In some embodiments, polynucleotide are
additionally cleaved using other means (e.g., restriction
digestion, mechanical cleavage) before or after cleavage and
labeling at an abasic site using the methods of the invention.
[0105] 2. Cleaving the Backbone at the Abasic Site of the
Polynucleotide Comprising an Abasic Site and Labeling at the Abasic
Site
[0106] The backbone of the polynucleotide is cleaved at the abasic
site, and the abasic site is labeled, whereby labeled
polynucleotide fragments are generated. It is understood that
cleavage of the backbone and labeling can be performed in any
order, or simultaneously. For convenience, however, these reactions
are described as separate steps.
[0107] i. Cleaving the Backbone at the Abasic Site of the
Polynucleotide Comprising an Abasic Site
[0108] Following generation of an abasic site, the backbone of the
polynucleotide is cleaved at the abasic site (i.e., the site
generated following cleavage of the base portion of the methylated
nucleotide) with an agent capable of effecting cleavage of the
backbone at the abasic site. Cleavage at the backbone (also termed
"fragmentation") results in at least two fragments (depending on
the number of abasic sites present in the polynucleotide comprising
an abasic site, and the extent of cleavage).
[0109] Suitable agents (for example, an enzyme, a chemical and/or
reaction conditions such as heat) capable of cleavage of the
backbone at an abasic site are well known in the art, and include:
heat treatment and/or chemical treatment (including basic
conditions, acidic conditions, alkylating conditions, or amine
mediated cleavage of abasic sites, (see e.g., co-pending co-owned
U.S. patent application Ser. No. 10/441,663; McHugh and Knowland,
Nucl. Acids Res. (1995) 23(10):1664-1670; Bioorgan. Med. Chem
(1991) 7:2351; Sugiyama, Chem. Res. Toxicol. (1994) 7: 673-83;
Horn, Nucl. Acids. Res., (1988) 16:11559-71), and use of enzymes
that catalyze cleavage of polynucleotides at abasic sites, for
example AP endonucleases (also called "apurinic, apyrimidinic
endonucleases") (e.g., E. coli Endonuclease IV, available from
Epicentre Tech., Inc, Madison Wis.), E. coli endonuclease III or
endonuclease IV, E. coli exonuclease III in the presence of calcium
ions. See, e.g. co-pending co-owned U.S. patent application Ser.
No. 10/441,663; Lindahl, PNAS (1974) 71(9):3649-3653; Jendrisak,
U.S. Pat. No. 6,190,865 B1; Shida, Nucleic Acids Res. (1996)
24(22):4572-76; Srivastava, J. Biol Chem. (1998) 273(13):21203-209;
Carey, Biochem. (1999) 38:16553-60; Chem Res Toxicol (1994)
7:673-683. As used herein "agent" encompasses reaction conditions
such as heat. In one embodiment, the AP endonuclease, E. coli
endonuclease IV, is used to cleave the phosphodiester backbone at
an abasic site. In another embodiment, cleavage is with an amine,
such as N,N'-dimethylethylenediamine. See, e.g. McHugh and
Knowland, supra.
[0110] Generally, cleavage is between the nucleotide immediately 5'
to the abasic residue and the abasic residue, or between the
nucleotide immediately 3' to the abasic residue and the abasic
residue (though, as explained herein, 5' or 3' cleavage of the
phosphodiester backbone may or may not result in retention of the
phosphate group 5' or 3' to the abasic site, respectively,
depending on the fragmentation agent used). As is well known in the
art, cleavage can be 5' to the abasic site (such as endonuclease IV
treatment which generally results in cleavage of the backbone at a
location immediately 5' to the abasic site between the 5'-phosphate
group of the abasic residue and the deoxyribose ring of the
adjacent nucleotide, generating a free 3' hydroxyl group on the
adjacent nucleotide), such that an abasic site is located at the 5'
end of the resulting fragment. Cleavage can also be 3' to the
abasic site (e.g., cleavage between the deoxyribose ring and
3'-phosphate group of the abasic residue and the deoxyribose ring
of the adjacent nucleotide, generating a free 5' phosphate group on
the deoxyribose ring of the adjacent nucleotide), such that an
abasic site is located at the 3' end of the resulting fragment.
Treatment under basic conditions or with amines (such as
N,N'-dimethylethylenediamine) results in cleavage of the
phosphodiester backbone immediately 3' to the abasic site. In
addition, more complex forms of cleavage are also possible, for
example, cleavage such that cleavage of the phosphodiester backbone
and cleavage of (a portion of) the abasic site results. For
example, under certain conditions, cleavage using chemical
treatment and/or thermal treatment may comprise a
.beta.-elimination step which results in cleavage of a bond between
the abasic site deoxyribose ring and its 3' phosphate, generating a
reactive .alpha.,.beta.-unsaturated aldehyde which can be labeled
or can undergo further cleavage and cyclization reactions. See,
e.g. Sugiyama, Chem. Res. Toxicol. (1994) 7: 673-83; Horn, Nucl.
Acids. Res., (1988) 16:11559-71. It is understood that more than
one method of cleavage can be used, including two or more different
methods which result in multiple, different types of cleavage
products (e.g., fragments comprising an abasic site at the 3' end,
and fragments comprising an abasic site at the 5' end).
[0111] Generally, cleavage of the backbone at an abasic site is
general, specific or selective cleavage (in the sense that the
agent (such as an enzyme) capable of cleaving the backbone at an
abasic site specifically or selectively cleaves the backbone at an
abasic site), whereby greater than about 98%, about 95%, about 90%,
about 85%, or about 80% of the cleavage is at an abasic site.
However, extent of cleavage can be less. Thus, reference to
specific cleavage is exemplary. In some embodiments, specific or
selective cleavage is desirable for control of the fragment size in
the methods of generating labeled polynucleotide fragments of the
invention. In some embodiments, reaction conditions can be selected
such that the cleavage reaction is performed in the presence of a
large excess of reagents and allowed to run to completion. In other
embodiments, extent of cleavage can be less, such that
polynucleotide fragments are generated comprising an abasic site at
an end and an abasic site(s) within or internal to the
polynucleotide fragment (i.e., not at an end). As disclosed herein,
polynucleotide fragments comprising internal abasic sites are
useful e.g., in embodiments involving immobilization of a labeled
polynucleotide (wherein one abasic site is used for immobilization
and another abasic site(s) are labeled at an abasic site).
[0112] As noted herein, the approximate or average size of the
fragments (following cleavage of an abasic site, and cleavage of
the backbone at the abasic site as described herein) is controlled
by variables known in the art, including: frequency of cleavable
nucleotides in the polynucleotide (in some embodiments involving
methylation analysis, frequency of methylated nucleotides), or
other measures of nucleotide content of a sequence, such as average
G-C content), length of the polynucleotide, and the reaction
conditions used during generation of abasic site and cleavage of
the backbone at the abasic site. In some embodiments,
polynucleotide are additionally cleaved using other means (e.g.,
restriction digestion, mechanical cleavage) before or after
cleavage and labeling at an abasic site using the methods of the
invention. Generally, suitable fragment sizes are about 5, 10, 15,
20, 25, 30, 40, 50, 65, 75, 85, 100, 123, 150, 175, 200, 225, 250,
300, 350, 400, 450, 500, 550, 600, 650 or more, such as 1 kb, 2 kb,
3 kb, 4 kb, 5 kb or more nucleotides in length. In some
embodiments, the fragment is about 200 nucleotides, about 100
nucleotides, or about 50 nucleotides in length. In another
embodiment, the size of a population of fragments is about 50 to
200 nucleotides. It is understood that the fragment size may be
approximate, particularly when populations of fragments are
generated, because the frequency and spacing of abasic sites (which
relates to the fragment size following cleavage) will vary from
template to template and also between copies of the same template,
due to representation of the methylated nucleotide, reactions
conditions selected for generation of abasic sites, and reaction
conditions selected for fragmentation. Thus, in some embodiments,
fragments generated from same starting material (such as a single
polynucleotide template) may have different (and/or overlapping)
sequence, while still having the same approximate size or size
range.
[0113] Following cleavage of the polynucleotide backbone at the
abasic site, every fragment will comprise one abasic site (if
cleavage is completely efficient), except for either the 5'- or
3'-most fragment, which may lack an abasic site depending on the
cleavage agent. If the cleavage is 5' to the abasic site, the 5'
most fragment will not comprise an abasic site. If cleavage is 3'
to the abasic site, the 3' most fragment will not comprise an
abasic site.
[0114] It is understood that the preceding disclosure regarding
cleavage of the backbone at the abasic site is applicable to
embodiments involving cleavage of the backbone at the abasic site
wherein the abasic site was generated by cleavage of a cleavable
canonical nucleotide with an agent capable of cleaving a base
portion of a canonical nucleotide, as described infra.
[0115] ii. Labeling the Abasic Site and Detection
[0116] The abasic site is labeled, whereby a polynucleotide (or
polynucleotide fragment) comprising a label is generated. In some
embodiments, a polynucleotide fragment comprising an abasic site is
contacted with an agent capable of labeling at the abasic site;
whereby labeled fragments of the polynucleotide are generated. As
used herein, a "label" (interchangeably called a "detectable
moiety") is attached to or associated with a polynucleotide, such
that the polynucleotide comprising an abasic site is attached to or
associated with a label.
[0117] Thus, in some embodiments, the label attaches to or
associates with a chemical structure remaining following treatment
of a methylated nucleotide (present in a polynucleotide chain) with
an agent (e.g., an enzyme, or acidic conditions, or a chemical
reagent) capable of effecting cleavage of a base portion of the
methylated nucleotide. In embodiments involving fragmentation, the
label attaches to or associates with any chemical structure
remaining following treatment of the methylated nucleotide (present
in a polynucleotide chain) with an agent (e.g., an enzyme, or
acidic conditions, or a chemical reagent) capable of effecting
cleavage of a base portion of the methylated nucleotide (in
embodiments involving cleavage of a methylated nucleotide), and
following treatment with an agent capable of cleaving the backbone
at the abasic site. In one embodiment, the label covalently bonds
with a reactive aldehyde form of a hemiacetal ring in an abasic
site. In some embodiments, labeling "at" an abasic site encompasses
labels that bind to an abasic site, but do not bind to the intact
(uncleaved) methylated nucleotide (in embodiments involving
cleavage of a methylated nucleotide) whether incorporated or
present as a single methylated nucleotide). In some embodiments,
labeling "at" an abasic site specifically excludes labels that
attach (e.g., covalently bind) to a phosphate group of a nucleotide
(or polynucleotide) or a phosphate group of an abasic site. In some
embodiments, labeling "at" an abasic site specifically excludes
labels that attach or associate at the 3' position of the sugar. In
still other embodiments, labeling "at" an abasic site specifically
excludes a label comprised of a phosphine. As made clear from the
disclosure herein, "label" refers to any component of a labeling
system.
[0118] It is understood that cleavage of the phosphodiester
backbone at the abasic site, and labeling at an abasic site can be
performed in any order, or simultaneously.
[0119] The label can be detectable, or the label can be indirectly
detected, for example as when the label (attached at an abasic
residue) is covalently or non-covalently associated with another
moiety which is itself detected. For example, biotin can be
attached to the label capable of associating with the abasic site.
In another example, an antibody (that can be detectably labeled)
binds the label that is attached at the abasic site. In some
embodiments, the label comprises an organic molecule, a hapten, or
a particle (such as a polystyrene bead). In some embodiments, the
label is detected using antibody binding, biotin binding, or via
fluorescence or enzyme activity. In some embodiments, the
detectable signal is amplified.
[0120] Generally, labeling at an abasic site is general, specific,
or selective labeling (in the sense that the agent capable of
labeling at an abasic site specifically or selectively labels the
abasic site), whereby greater than about 98%, about 95%, about 93%,
about 90%, about 85%, or about 80% of the labels bind abasic sites.
However, extent of labeling can be less. Thus, reference to
specific labeling is exemplary. In some embodiments, reaction
conditions are selected such that the reaction in which the abasic
site(s) are labeled can run to completion.
[0121] In some embodiments, labeled polynucleotide fragments are
produced which each comprise a single label (to the extent that
cleavage of the phosphodiester backbone is generally complete, in
the sense that many or essentially all of the polynucleotide
fragments comprise a single abasic site). This aspect is useful in
quantitating level of hybridization, because signal is proportional
to number of bound fragments, and does not relate to the length of
the hybridizing fragment or the number of labels per fragment.
Thus, hybridization intensity can generally be directly compared,
regardless of fragment length. In another embodiment, labeled
fragments are produced which comprise a labeled abasic site at an
end (such as the 3' end and/or the 5' end) and a labeled internal
abasic site.
[0122] Methods and reaction conditions for labeling abasic sites
are known in the art. See, e.g., co-pending co-owned U.S. patent
application Ser. No. 10/441,663. For example, a common functional
group exposed in an abasic site (and therefore suitable for use in
labeling) is the highly reactive aldehyde form of the hemiacetal
ring which can be covalently attached to or noncovalently
associated with a label using reaction conditions that are known in
the art. Many labels comprise substituted hydrazines or
hydroxylamines which readily form imine bonds with aldehydes, for
example, 5-(((2-(carbohydrazino)-methyl)thio)acetyl)aminof-
luorescein, aminooxyacetyl hydrazide (FARP). See Makrigiorgos, PCT
Publication No. WO 00/39345. The stable oxime formed by this
compound can be detected directly by fluorescence or the signal can
be amplified using an antibody-enzyme conjugate. See, e.g.,
Srivastava, J. Biol. Chem. (1998) 273(33): 21203-209; Makrigiorgos,
Int J. Radiat. Biol. (1998) 74(1):99-109; Makrigiorgos, U.S. Pat.
No. 6,174,680 B1; Makrigiorgos, WO 00/39345. Suitable sidechains
(present on the substrate) to react with the aldehyde (of the
abasic site) include at least the following: substituted
hydrazines, hydrazides, or hydroxylamines (which readily form imine
bonds with aldehydes), and the related semicarbazide and
thiosemicarbazide groups, and other amines which can form stable
carbon-nitrogen double bonds, that can catalyze simultaneous
cleavage and binding (see Horn, Nucl. Acids. Res., (1988)
16:11559-71), or can be coupled to form stable conjugates, e.g. by
reductive amination. Other methods for attaching a reactive group
present in an abasic site to a reactive group present on a label
are known in the art. In another example, the abasic site may be
chemically modified, then the modified abasic site covalently
attached to or non-covalently associated with a suitable reactive
group on a substrate. For example, the aldehyde (in the abasic
site) can be oxidized or reduced (using methods known in the art),
then covalently immobilized to a substrate using, e.g., reductive
amination or various oxidative processes.
[0123] Other suitable reagents are known in the art, e.g.,
fluorescein aldehyde reagents. See, e.g., Boturyn (1999) Chem. Res.
Toxicol. 12:476-482. See, also, Adamczyk (1998) Bioorg. Med. Chem.
Lett. 8(24):3599-3602; Adamczyk (1999) Org. Lett. 1(5):779-781; Kow
(2000) Methods 22(2):164-169; Molecular Probes Handbook, Section
3.2 (www.probes.com). For example, detectable moieties comprising
aminooxy groups can be used. See, Boturyn, supra. The aminoooxy
group readily reacts with the highly reactive aldehyde form of the
hemiacetal ring of an abasic site. In one embodiment, the label
comprising an aminooxy reactive group is
N-(aminooxyacetyl)-N'-(D-biotinoyl) hydrazine, trifluoroacetic acid
salt (ARP) (available from Molecular Probes, Eugene Oreg., catalog
No. A-10550). See, e.g., Kubo et al., Biochem 31:3703-3708 (1992);
Ide et al., Biochem. 32:8276-8283 (1993).
[0124] In yet another example, labels comprising a hydrazide linker
can be converted to an aminooxy (interchangeably termed
"hydroxylamine") derivative, then used to label abasic sites as
described herein. In one embodiment, the label comprises an
aminooxy derivatized Alexa Fluor 555 reagent as disclosed in
co-pending U.S. patent application Ser. No. 10/441,663, Use of the
aminooxy-derivatized Alexa Fluor 555 resulted in greater labeling
efficiency, as well as increased fluorescence as compared to
labeling with unmodified Alexa Fluor 555 hydrazide (Order No.
A-20501, Molecule Probes, Eugene Oreg.).
[0125] In another example, the abasic site may be chemically
modified (before, during or after cleavage of the phosphodiester
backbone as described herein), then the modified abasic site
detected directly or indirectly. For example, fluorescent
cadaverine can be incorporated into an abasic site as described in
Horn (Nucl. Acids. Res., (1988) 16:11559-71). In another example,
the abasic site may be chemically modified by reaction with NHBA
(0-4-nitrobenzyl hydroxylamine), then the NBHA-modified abasic site
is detected with an antibody that specifically binds to the
NBHA-modified abasic sites. See Kow et al, PCT Publication No. WO
92/07951 (1992).
[0126] In another example, the abasic site may be labeled with an
antibody (such as a monoclonal or polyclonal antibody or antigen
binding fragment). Methods for detecting specific antibody binding
are well known in the art.
[0127] In another example, the aldehyde and/or hemiacetal ring may
itself be detected, as when, for example, detectable signal is
generated using chemical or electrochemical reactions specific to
those chemical structures, including for example, oxidation
reactions, enzymes with dehydrogenase or oxidase activity, and the
like. In another example, many aldehydes are substrates for
enzymes, such that a detectable product is generated in the
presence of the aldehyde. For example, dehydrogenases typically
couple oxidation of an aldehyde with reduction of NAD+ which can be
detected spectrophotometrically. In another example, glucose
oxidases generate hydrogen peroxide in the presence of sugar
aldehydes. Hydrogen peroxide is readily detectable by coupling to
horseradish peroxidase with suitable substrates. Thus, the
invention provides methods for detecting an abasic site.
[0128] Methods of signal detection are known in the art: Signal
detection may be visual or utilize a suitable instrument
appropriate to the particular label used, such as a spectrometer,
fluorimeter, or microscope. For example, where the label is a
radioisotope, detection can be achieved using, for example, a
scintillation counter, or photographic film as in autoradiography.
Where a fluorescent label is used, detection may be by exciting the
fluorochrome with the appropriate wavelength of light and detecting
the resulting fluorescence, such as by microscopy, visual
inspection or photographic film, fluorometer, CCD cameras, scanner
and the like. Where enzymatic labels are used, detection may be by
providing appropriate substrates for the enzyme and detecting the
resulting reaction product. For example, many substrates of
horseradish peroxidase, such as o-phenylenediamine, give colored
products. Simple colorimetric labels can usually be detected by
visual observation of the color associated with the label; for
example, conjugated colloidal gold is often pink to reddish, and
beads appear the color of the bead. Instruments suitable for high
sensitivity detection are known in the art.
[0129] It is understood that the polynucleotide or polynucleotide
fragments can be additionally labeled using other methods known in
the art.
[0130] Labeled polynucleotide fragments can be immobilized to a
substrate, as described herein.
[0131] It is understood that the preceding disclosure regarding
labeling at the abasic site is applicable to embodiments involving
labeling at the abasic site wherein the abasic site was generated
by cleavage of a cleavable canonical nucleotide with an agent
capable of cleaving a base portion of a canonical nucleotide, as
described infra.
[0132] B. Methods for Labeling a Polynucleotide Comprising a
Methylated Nucleotide
[0133] The invention provides methods for generating labeled
nucleic acid(s).
[0134] Generally, the methods comprise use of an agent (such as an
enzyme) that cleaves a base portion from a methylated nucleotide
(such as 5-methylcytosine), whereby an abasic site is generated;
and labeling at the abasic site, whereby labeled polynucleotides
are generated. Generally, the polynucleotide comprising a
methylated nucleotide (in some embodiments, suspected of comprising
a methylated nucleotide) is labeled at the abasic site (which is
generated by cleavage of a base portion of the methylated
nucleotide). The methods of the invention generate labeled
polynucleotides which are useful for, e.g., hybridization to a
microarray and other uses described herein. The methods of the
invention are suitable for multiplexing.
[0135] The methods involve the following steps: (a) contacting a
polynucleotide comprising (in some embodiments, suspected of
comprising) a methylated nucleotide with an agent capable of
cleaving a base portion of the methylated nucleotide (i.e.,
cleaving a base portion of the methylated nucleotide), whereby an
abasic site is generated; and (b) contacting the polynucleotide
comprising the abasic site with an agent capable of labeling the
abasic site (i.e., labeling the abasic site), whereby labeled
polynucleotides are generated.
[0136] It is understood that labeled polynucleotides may be cleaved
using other means (i.e. other means than cleavage at an abasic site
as described herein, e.g., restriction digestion, mechanical
cleavage) before or after labeling at an abasic site using the
methods of the invention. In some embodiments, the methods
involving labeling of a polynucleotide comprising a methylated
nucleotide are suitable for multiplex analysis involving
hybridization to an array.
[0137] 1. Cleaving a Base Portion of a Methylated Nucleotide to
Create an Abasic Site
[0138] The polynucleotide comprising a methylated nucleotide (in
some embodiments, suspected of comprising a methylated nucleotide)
is treated with an agent, such as an enzyme, capable of generally,
specifically, or selectively cleaving a base portion of the
methylated deoxyribonucleoside to create an abasic site, as
described herein.
[0139] 2. Labeling the Abasic Site and Detection
[0140] The abasic site is labeled, whereby a polynucleotide
comprising a label is generated, as described herein.
[0141] C. Methods for Fragmenting a Polynucleotide Comprising a
Methylated Nucleotide
[0142] The methods involve the following steps: (a) contacting a
polynucleotide comprising (in some embodiments, suspected of
comprising) a methylated nucleotide with an agent capable of
cleaving a base portion of the methylated nucleotide (i.e.,
cleaving a base portion of the methylated nucleotide), whereby an
abasic site is generated; and (b) cleaving the backbone of the
polynucleotide comprising the abasic site at the abasic site,
whereby polynucleotide fragments are generated.
[0143] 1. Cleaving a Base Portion of a Methylated Nucleotide to
Create an Abasic Site
[0144] The polynucleotide comprising a methylated nucleotide (in
some embodiments, suspected of comprising a methylated nucleotide)
is treated with an agent, such as an enzyme, capable of generally,
specifically, or selectively cleaving a base portion of the
methylated deoxyribonucleoside to create an abasic site, as
described herein.
[0145] 2. Cleaving the Backbone at the Abasic Site of the
Polynucleotide Comprising an Abasic Site and Labeling at the Abasic
Site
[0146] The backbone of the polynucleotide is cleaved at the abasic
site, and the abasic site is labeled, whereby polynucleotide
fragments are generated, as described herein.
[0147] D. Methods for Labeling and Fragmenting a Polynucleotide
Comprising a Methylated Nucleotide Using an Enzyme that Cleaves a
Base Portion of a Nonmethylated Nucleotide
[0148] In another aspect, the invention comprises use of an enzyme
(such as cytosine deaminase in conjunction with uracil N
deglycosylase (UNG)) that cleaves a base portion from an
unmethylated nucleotide, whereby an abasic site is generated,
wherein the enzyme is not capable of cleaving a methylated
nucleotide; cleavage of the phosphodiester backbone of the
polynucleotide comprising the abasic site at the abasic site; and
labeling at the abasic site, whereby labeled polynucleotide
fragments are generated. Generally, the polynucleotide comprising a
methylated nucleotide (in some embodiments, suspected of comprising
a methylated nucleotide) is fragmented and labeled at the abasic
site (which is generated by cleavage of a base portion of the
non-methylated nucleotide). Thus, the methods of the invention are
useful for, e.g., detecting methylation (including presence and/or
absence and/or quantity or level of methylation), identifying
methylated polynucleotide sequences, isolating methylated
polynucleotide sequences, identifying and/or isolating methylatable
polynucleotide sequences, and other applications as described
herein. The methods of the invention generate labeled
polynucleotide fragments which are useful for, e.g., hybridization
to a microarray and other uses described herein.
[0149] The methods comprise the following steps: (a) contacting a
polynucleotide comprising (in some embodiments, suspected of
comprising) a methylated nucleotide with an enzyme capable of
cleaving a base portion of an unmethylated nucleotide (i.e.,
cleaving a base portion of the unmethylated nucleotide), whereby an
abasic site is generated, wherein the enzyme is not capable of
cleaving a methylated nucleotide; (b) cleaving the backbone of the
polynucleotide comprising the abasic site at the abasic site; and
(c) contacting the polynucleotide comprising the abasic site with
an agent capable of labeling the abasic site (i.e., labeling the
abasic site), whereby labeled polynucleotide fragments are
generated. In some embodiments, the enzyme is cytosine deaminase in
conjunction with uracil DNA glycosylase.
[0150] 1. Cleaving a Base Portion of an Unmethylated Nucleotide
[0151] In another aspect, the invention comprises use of an enzyme
(such as cytosine deaminase in conjunction with uracil DNA
glycosylase) that cleaves a base portion of an unmethylated
nucleotide, wherein the enzyme is not capable of cleaving a
methylated nucleotide; cleavage of the phosphodiester backbone of
the polynucleotide comprising the abasic site at the abasic site;
and labeling at the abasic site, whereby labeled polynucleotide
fragments are generated.
[0152] Generally, the methods involve the following steps: (a)
contacting a polynucleotide comprising (in some embodiments,
suspected of comprising) a methylated nucleotide with an agent
(such as an enzyme) capable of cleaving a base portion of an
unmethylated nucleotide (i.e., cleaving a base portion of the
unmethylated nucleotide), whereby an abasic site is generated,
wherein the agent (such as an enzyme) is not capable of cleaving
the methylated nucleotide; (b) cleaving the backbone of the
polynucleotide comprising the abasic site at the abasic site; and
(c) contacting the polynucleotide comprising the abasic site with
an agent capable of labeling the abasic site (i.e., labeling the
abasic site), whereby labeled polynucleotide fragments are
generated.
[0153] In some embodiments, the enzyme is cytosine deaminase. See
Sohail et al, NAR 2003, 31: 2990-94. Cytosine deaminase catalyzes
the deamination of cytosine, such that dUTP is generated. Cleavage
of a base portion of dUTP is necessary to generate the abasic site.
Thus, the invention encompasses use of (a) an agent (such as
cytosine deaminase) that modifies a nucleotide (such as dCTP),
whereby dUTP is generated, in conjunction with (b) an agent (such
as an enzyme, such as UNG) that cleaves a base portion of dUTP,
whereby an abasic site is generated. Methods for cleaving a base
portion of dUTP are known in the art. See, e.g., Lindahl, PNAS
(1974) 71(9):3649-3653; Jendrisak, U.S. Pat. No. 6,190,865 B1; U.S.
Pat. No. 5,035,996; U.S. Pat. No. 5,418,149; Sartori et al (2002)
EMBO J 21:3182-3191. As used herein, "in conjunction" encompasses
simultaneous treatment (e.g., when cytosine deaminase and UNG
cleavage occurs in the same reaction mixture) and/or treatment at
different times (e.g., when cytosine deaminase and UNG treatment is
conducted sequentially).
[0154] In some embodiments, the agent that cleaves the base portion
of the unmethylated nucleotide is the same agent that cleaves a
phosphodiester backbone at the abasic site.
[0155] Generally, cleavage of the base portion of the unmethylated
nucleotides is general, specific or selective cleavage (in the
sense that the agent (such as an enzyme) capable of cleaving a base
portion of an unmethylated nucleotide generally, specifically or
selectively cleaves the base portion of a particular unmethylated
nucleotide), and generally, specifically and selectively does not
cleave the base portion of the methylated nucleotide, whereby about
any of 98%, 95%, 90%, 85%, or 80% of the base portions cleaved are
base portions of unmethylated nucleotides. However, extent of
cleavage can be less. Thus, reference to specific cleavage is
exemplary. In some embodiments, the methylated nucleotide is
5-methylcytosine and the unmethylated nucleotide is cytosine.
[0156] As noted herein, for convenience, cleavage of a base portion
of a unmethylated nucleotide (whereby an abasic site is generated)
has been described as a separate step. It is understood that this
step may be performed simultaneously with cleavage of the backbone
at an abasic site (fragmentation) and/or labeling at an abasic
site.
[0157] It is understood that the frequency (or spacing) of abasic
sites in the resulting polynucleotide comprising an abasic site
(following cleavage of a base portion of a unmethylated nucleotide,
and thus the average size of fragments generated using the methods
of the invention (i.e., following cleavage of a phosphodiester
backbone at an abasic site), is controlled by variables known in
the art, including: frequency of unmethylated nucleotide(s) in the
polynucleotide (or other measures of nucleotide content of a
sequence, such as average G-C content), length of the
polynucleotide comprising a unmethylated nucleotide, and the
reaction conditions used during generation of abasic site, as is
further discussed herein (such as presence of a methylation binding
protein, and/or presence of a DNA binding protein). In some
embodiments, polynucleotide are additionally cleaved using other
means (e.g., restriction digestion, mechanical cleavage) before or
after cleavage and labeling at an abasic site using the methods of
the invention.
[0158] It is understood that the methods involving cleavage of a
base portion of an unmethylated nucleotide relate to and comprise
use of a polynucleotide with native methylation status, i.e., a
polynucleotide in which methylation status or pattern exists in
vivo, as described herein. Specifically excluded are labeling
and/or fragmentation methods comprising synthesis of a
polynucleotide comprising a non-canonical nucleotide as disclosed
in co-pending co-owned U.S. patent application Ser. No. 10/441,663
(publication no. 2004/0005614).
[0159] Generally, the methods involving cleavage of a nonmethylated
nucleotide (but not cleavage of the corresponding methylated
nucleotide) are suitable for use with polynucleotide templates that
are heavily methylated (hypermethylated), though other
polynucleotide templates are suitable for use in the methods of the
invention. Methods involving cleavage in the presence of methyl
binding proteins and/or DNA binding proteins are further described
herein.
[0160] In some embodiments, the polynucleotide comprising
methylated nucleotide is contacted with a methyl binding agent,
such as a methyl binding antibody, and/or a methyl binding protein,
prior to and/or during cleavage of the base portion of the
unmethylated nucleotide. Binding with a methyl binding agent
protects the portion of the polynucleotide bound from labeling and
fragmentation (including cleavage of a nomnethylated nucleotide).
For example, when using an enzyme for cleavage of the base, binding
of the enzyme to a methylated nucleotide base or activity of the
enzyme towards a methylated nucleotide, may be prevented by binding
to the methyl binding agent. Thus, the methods generally produce
polynucleotide fragments comprising a methylated nucleotide that
are suitable for further analysis, such as by hybridization to a
microarray. The methods are generally suitable, e.g., for the
analysis of hypermethylated samples, where methods involving
cleavage at a methylated nucleotide may result in excessive
fragmentation. In some embodiments, the methods are useful for
selectively fragmenting a non-hypermethylated sequence in the
polynucleotide of interest, such that hypermethylated sequences may
be isolated from the reaction mixture and further analyzed
(including sequencing and/or cloning and/or hybridization to a
microarray).
[0161] Antibodies that bind methylated nucleotides are known in the
art. See, e.g., U.S. Patent Publication No. 2002/0197639; Erlanger
and Beiser (PNAS, 52:68, 1964); Sano et al., (Biochemica et
Biophysica Acta, 951:157, 1988); PCT Publication No. WO 99/10540
published on Mar. 4, 1999; Mizugaki et al (1996) Biol Pharm. Bull.
19:1537-40 (describing antibodies that recognize 5-methylcytosine)
and Tohuku J. Exp Med. 1986, 149(2):151-161. Methyl binding
proteins are also known in the art. See, e.g, Ballestar et al
(2001) Eur J Biochem. 268: 1-6. As is well known in the art, a
methyl binding protein (also termed methylation binding protein or
MBP) encompasses proteins that bind methylated nucleotides as well
as proteins that bind a region including a methylated
nucleotide.
[0162] In other embodiments, the polynucleotide comprising a
methylated nucleotide is contacted with a polynucleotide (e.g. DNA)
binding protein, such as a transcription factor, regulatory
protein, and other DNA binding proteins, prior to cleavage of the
base portion of the unmethylated nucleotide. The polynucleotide
binding protein, e.g. a transcription factor, protects the bound
portion of the polynucleotide from cleavage, thus generating a
labeled and fragmented polynucleotide comprising the binding site
of the particular DNA binding protein.
[0163] In some embodiments, methylation status may be compared in
the presence or absence or methylation binding agents and/or other
polynucleotide binding proteins. As further described herein,
comparison of methylation status is useful to compare samples from,
e.g., different stages of development, and/or different disease
states (such as a control (normal) sample and a sample from
diseased tissue).
[0164] 2. Cleaving the Backbone at the Abasic Site of the
Polynucleotide Comprising an Abasic Site
[0165] Following generation of an abasic site, the backbone of the
polynucleotide is cleaved at the abasic site (i.e., the site
generated following cleavage of the base portion of the canonical
nucleotide) with an agent capable of effecting cleavage of the
backbone at the abasic site, as described herein.
[0166] 3. Labeling the Abasic Site and Detection
[0167] The abasic site is labeled, whereby a polynucleotide (or
polynucleotide fragment) comprising a label is generated as
described herein. In some embodiments, a polynucleotide fragment
comprising an abasic site is contacted with an agent capable of
labeling at the abasic site; whereby labeled fragments of the
polynucleotide are generated as described herein.
[0168] E. Applications Using the Labeling and/or Fragmentation
Methods for Analysis of DNA Methylation
[0169] The methods and compositions of the invention can be used
for a variety of purposes. For purposes of illustration, methods of
producing a hybridization probe or target characterizing and/or
quantitating nucleic acid, preparing a subtractive hybridization
probe, detection (using the hybridization probe), and determining
methylation status, using the labeled and/or fragmented nucleic
acids generated by the methods of the invention, are described.
[0170] 1. Methods for Characterizing Methylated Polynucleotides
[0171] The labeled and/or fragmented nucleic acids obtained by the
methods of the invention are amenable to further
characterization.
[0172] The labeled and/or fragmented nucleic acids (i.e., products
of any of the methods for labeling and/or fragmenting a
polynucleotide comprising a methylated nucleotide described
herein), can be analyzed using, for example, probe hybridization
techniques known in the art, such as Southern and Northern
blotting, and hybridizing to probe arrays. They can also be
analyzed by electrophoresis-based methods, such as differential
display and size characterization, which are known in the art,
e.g., capillary electrophoresis and gel electrophoresis using a
sequencing gel.
[0173] In one embodiment, the methods of the invention are utilized
to generate labeled and/or fragmented nucleic acids, and analyze
the labeled and/or fragmented nucleic acids by contact with a
probe.
[0174] In one embodiment, the methods of the invention are utilized
to generate labeled and/or fragmented nucleic acids which are
analyzed (for example, detection and/or quantification) by
contacting them with, for example, microarrays (of any suitable
substrate, which includes glass, chips, plastic), beads, or
particles, that comprise suitable probes such as cDNA and/or
oligonucleotide probes. Thus, the invention provides methods to
characterize (for example, detect and/or quantify and/or identify)
a labeled and/or fragmented nucleic acid by analyzing the labeled
products, for example, by hybridization of the labeled products to,
for example, probes immobilized at, for example, specific locations
on a solid or semi-solid substrate, probes immobilized on defined
particles (including beads, such as Bead Array, Illumina), or
probes immobilized on blots (such as a membrane), for example
arrays, or arrays of arrays. Immobilized probes include immobilized
probes generated by the methods described herein, and also include
at least the following microarrays: cDNA and synthetic
oligonucleotides, which can be synthesized directly on the
substrate. In some embodiments, the microarray is an Affymetrix
Gene Chip array, an Agilent oligonucleotide array, or Amersham
CodeLink array, or other high density or low density
oligonucleotide or cDNA array, including genome or focused arrays.
The identity of the probes provides characterization of the
sequence identity of the products, and thus by extrapolation can
also provide characterization of the identity of the polynucleotide
comprising a methylated nucleotide (termed "template") from which
the products were prepared. Thus, it is evident that
polynucleotides comprising a methylated nucleotide may be
identified using the methods of the invention.
[0175] Other methods of analyzing labeled products are known in the
art, such as, for example, by contacting them with a solution
comprising probes, followed by extraction of complexes comprising
the labeled products and probes from solution. The identity of the
probes provides characterization of the sequence identity of the
products, and thus by extrapolation can also provide
characterization of the identity of a template from which the
products were prepared. In addition, hybridization of the labeled
products is detectable, and the amount of labels that are detected
is proportional to the amount of the labeled products prepared from
a specific polynucleotide comprising a methylated nucleotide. This
measurement is useful for, for example, measuring the relative
amount (quantity, extent) of methylated species in a sample. The
amount of labeled products (as indicated by, for example,
detectable signal associated with the label) hybridized at defined
locations on an array is indicative of the detection and/or
quantification of the corresponding methylated polynucleotide
species in the sample.
[0176] In another aspect, the invention provides methods for
detecting methylated polynucleotides, including detecting presence
or absence or quantity (level and/or extent) of methylation in a
polynucleotide sample (which may comprise one or a multiplicity, a
large multiplicity or a very large multiplicity of polynucleotides
comprising a methylated nucleotide (in some embodiments, suspected
of comprising a methylated polynucleotide), including essentially
all genomic DNA. In some embodiments, presence or absence or
quantity of methylation is determined by hybridizing the labeled
and/or fragmented polynucleotides (generated using any of the
methods for labeling and/or fragmenting methylated polynucleotides
described herein) to a polynucleotide (probe) of defined sequence
(which may be immobilized, for example, on a microarray), as
described further herein. In some embodiments, the microarray is a
cDNA microarray. In some embodiments, the microarray is an
Affymetrix Gene Chip array, an Agilent oligonucleotide array, or
Amersham CodeLink array, or other high density or low density
oligonucleotide or cDNA array, including genome or focused arrays.
It is understood that amount of labeled and/or fragmented nucleic
acids produced (and thus the amount of product) may be determined
using quantitative and/or qualitative methods. Determining amount
of labeled and/or fragmented nucleic acids includes determining
whether labeled and/or fragmented nucleic acids are present or
absent. Thus, amount or extent of methylation includes information
relating to absence of methylation. "Absent" or "absence" of
product, and "lack of detection of product" as used herein includes
insignificant, or de minimus levels.
[0177] As described above, labeled and/or fragmented nucleic acids
can be detected and quantified by various methods, as described
herein and/or known in the art. Determination of the amount of
methylated polynucleotide of interest present in a sample, as
determined by quantifying products (for example labeled and/or
fragmented products) of the methods described herein, provides for
determination of the methylation status of the sample source. It is
understood that presence or absence or quantity of methylation may
be determined for one polynucleotide of interest, a multiplicity of
polynucleotides, a large multiplicity, or a very large multiplicity
of polynucleotides (including all or essentially all genomic DNA).
In some embodiments, presence or absence or quantity (extent or
pattern) of methylation is determined for a specific polynucleotide
sequence. In other embodiments, presence or absence or quantity is
determined for a multiplicity, a large multiplicity or a very large
multiplicity of polynucleotide sequences. Polynucleotide sequences
(for which methylation analysis is desired) may be known (e.g., a
particular locus, genomic region, CpG island, CpG dinucleotide,
etc.) or may be unknown or unidentified polynucleotide
sequences.
[0178] In another aspect, the invention provides methods for
isolating, enriching and/or identifying methylated polynucleotides.
The methods of the invention are utilized to generate labeled
and/or fragmented nucleic acids which are isolated, enriched and or
identified. In some embodiments, labeled and/or fragmented
polynucleotides are physically captured (for example, by binding to
probes in solution or microarray), and captured polynucleotide
products are isolated and or enriched. In other embodiments,
labeled and/or fragmented polynucleotide products are identified
via hybridization to a known probe or other means for
characterizing the sequence thereof.
[0179] 2. Comparison of Methylation Status
[0180] The labeled and/or fragmented nucleic acids generated
according to the methods of the invention are also suitable for
analysis for the detection of any alteration in methylation status
(e.g., presence or absence or quantity, extent and/or pattern) in
the template polynucleotide sequence (from which the labeled and/or
fragmented nucleic acids are synthesized), as compared to a
reference nucleic acid sequence which is identical to the template
nucleic acid sequence other than the alteration in methylation
status, if any.
[0181] Accordingly, the invention provides methods of comparing
methylation status in a sample, said method comprising: (a)
generating labeled and/or fragmented from at least one
polynucleotide template in the sample using any of the methods
described herein; and (b) determining amount of labeled
polynucleotide or fragments thereof of each polynucleotide
template, wherein each said amount is indicative of amount of each
polynucleotide template in the sample.
[0182] The methods are useful in a wide variety of molecular
diagnostics, and especially in the study of essentially any cell
(including a single cell) or cell population. A cell or cell
population (e.g. a tissue) may be from, for example, blood, brain,
spleen, bone, heart, vascular, lung, kidney, pituitary, endocrine
gland, embryonic cells, tumors, or the like. Comparison of
methylation status is also useful for comparing a control (normal)
sample to a test sample, including test samples collected at
different times, including before, after, and/or during
development, a treatment, and the like.
[0183] 3. Comparative Hybridization
[0184] In another aspect, the invention provides methods for
comparative hybridization (such as comparative genomic
hybridization), said method comprising: (a) preparing a first
population of labeled polynucleotides or fragments thereof from a
first template polynucleotide sample using any of the methods
described herein; (b) comparing hybridization of the first
population to at least one probe with hybridization of a second
population of labeled polynucleotides or fragments thereof. In
still other embodiments, the at least one probe is provided as a
microarray. In some embodiments, the first and second population
comprises detectably different labels. In other embodiments, the
second population of labeled polynucleotides, or fragments thereof,
are prepared from a second polynucleotide sample using any of the
methods described herein. In some embodiments, step (b) of
comparing comprises determining amount of said products, whereby
the amount of the first and second polynucleotide templates is
quantified.
[0185] In some embodiments, comparative hybridization comprises
preparing a first population of labeled polynucleotides (which can
be polynucleotide fragments) according to any of the methods
described herein. A second population of labeled polynucleotides
(to which the first population is desired to be compared) is
prepared from a second genomic DNA template. The first and second
population may be hybridized to a single array (in which case,
detectably different labels are generally used) or hybridized to
different arrays (in which case the labels may be the same or
different). Hybridization of the first and second populations is
detected and compared.
[0186] Methods for Labeling and Fragmenting Polynucleotides
Comprising a Cleavable Canonical Nucleotide, and Methods for
Labeling a Polynucleotide Comprising a Cleavable Canonical
Nucleotide
[0187] A. Methods for Labeling and Fragmenting Polynucleotides
Comprising a Cleavable Canonical Nucleotide
[0188] The invention provides methods for generating labeled
fragments of a polynucleotide comprising a cleavable canonical
nucleotide (interchangeably termed "canonical nucleotide"). In some
aspects, namely the RNA context, a canonical nucleotide includes a
nucleotide comprising the base uracil (U) (as well as respective
forms such as ribonucleoside, etc.).
[0189] The methods generally comprise cleavage of a base portion of
a canonical nucleotide present in a polynucleotide comprising the
canonical nucleotide with an agent capable of cleaving a base
portion of the canonical nucleotide; and cleavage of the
phosphodiester backbone of the polynucleotide comprising the abasic
site at the abasic site; and labeling at the abasic site, whereby
labeled nucleic acid fragments are generated. Generally, the
polynucleotide comprising a canonical nucleotide is fragmented and
labeled at the abasic site (generated by cleavage of a base portion
of a canonical nucleotide). Thus, the frequency of abasic sites
generated by cleavage of the base portion of the canonical
nucleotide generally relates to and determines the size range of
the labeled fragments produced from the polynucleotide. The methods
of the invention generate labeled nucleic acid fragments, which are
useful for, for example, hybridization to a microarray and other
uses described herein.
[0190] The methods involve the following steps: (a) contacting a
polynucleotide comprising a canonical nucleotide with an agent
capable of cleaving a base portion of the canonical nucleotide
(i.e., cleaving a base portion of the canonical nucleotide),
whereby an abasic site is created; (b) cleaving the backbone of the
polynucleotide comprising the abasic site at the abasic site; and
(c) contacting the polynucleotide comprising the abasic site with
an agent capable of labeling the abasic site (i.e., labeling the
abasic site), whereby labeled polynucleotide fragments are
generated.
[0191] For simplicity, individual steps of the labeling and
fragmentation method are discussed below. It is understood,
however, that the steps may be performed simultaneously and/or in
varied order, as discussed herein.
[0192] In some embodiments, an abasic site is generated at about
every 5, 10, 15, 20, 25, 30, 40, 50, 65, 75, 85, 100, 123, 150,
175, 200, 225, 250,300, 350, 400, 450, 500, 550, 600, 650 or more
nucleotides apart. In one embodiment, the abasic site is generated
about every 200 nucleotides, about every 100 nucleotide, or about
every 50 nucleotide. In another embodiment, the abasic site is
generated about every 50 to about 200 nucleotides.
[0193] The frequency (or spacing) of abasic sites in the resulting
polynucleotide comprising an abasic site (following cleavage of a
base portion of a canonical nucleotide, and thus the average size
of fragments generated using the methods of the invention (i.e.,
following cleavage of a phosphodiester backbone at an abasic site),
is controlled by variables known in the art, including: frequency
of canonical nucleotide(s) in the polynucleotide (or other measures
of nucleotide content of a sequence, such as average G-C
content),and the reaction conditions used during generation of
abasic site, as is further discussed herein. The reaction
conditions can be empirically determined, for example, by assessing
average fragment size generated using the methods of the invention
taught herein. In some embodiments, about any of 1%, 2%, 3%, 4%,
5%, 6%, 7%, 8%, 9 %,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more canonical
bases are cleaved.
[0194] The polynucleotide comprising a canonical nucleotide may be
any template from which labeled polynucleotide fragments are
desired to be produced, including double-stranded, partially
double-stranded, and single-stranded nucleic acids from any source
in purified or unpurified form, which can be DNA (dsDNA and ssDNA)
or RNA, including tRNA, mRNA, rRNA, mitochondrial DNA and RNA,
chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof,
genes, chromosomes, plasmids, the genomes of biological material
such as microorganisms, e.g., bacteria, yeasts, viruses, viroids,
molds, fungi, plants, animals, humans, and fragments thereof.
Obtaining and purifying nucleic acids use standard techniques in
the art. RNAs can be obtained and purified using standard
techniques in the art. A DNA template (including genomic DNA
template) can be transcribed into RNA form, which can be achieved
using methods disclosed in Kurn, U.S. Pat. No. 6,251,639 B1, and by
other techniques (such as expression systems) known in the art. RNA
copies of genomic DNA would generally include untranscribed
sequences generally not found in mRNA, such as introns, regulatory
and control elements, etc. DNA copies of an RNA template can be
synthesized using methods described in Kum, U.S. Patent Publication
No. 2003/0087251 A1 or other techniques known in the art).
Synthesis of polynucleotide from a DNA-RNA hybrid can be
accomplished by denaturation of the hybrid to obtain a ssDNA and/or
RNA, cleavage with an agent capable of cleaving RNA from an RNA/DNA
hybrid, and other methods known in the art. The template can be
only a minor fraction of a complex mixture such as a biological
sample and can be obtained from various biological materials by
procedures well known in the art. The template can be known or
unknown and may contain more than one desired specific nucleic acid
sequence of interest, each of which may be the same or different
from each other. The template DNA can be a sub-population of
nucleic acids, for example, a subtractive hybridization probe,
total genomic DNA, restriction fragments, a cDNA library, cDNA
prepared from total mRNA, a cloned library, or amplification
products of any of the templates described herein. In some cases,
the initial step of the synthesis of the complement of a portion of
a template nucleic acid sequence is template denaturation. The
denaturation step may be thermal denaturation or any other method
known in the art, such as alkali treatment.
[0195] For simplicity, the polynucleotide comprising a canonical
nucleotide is described as a single nucleic acid. It is understood
that the polynucleotide can be a single polynucleotide, or a
population of polynucleotides (from a few to a multiplicity to a
very large multiplicity of polynucleotides). It is further
understood that a polynucleotide comprising a canonical nucleotide
can be a multiplicity (from small to very large) of different
polynucleotide molecules. Such populations can be related in
sequence (e.g., member of a gene family or superfamily) or
extremely diverse in sequence (e.g., generated from all mRNA,
generated from all genomic DNA, etc.). Polynucleotides can also
correspond to single sequence (which can be part or all of a known
gene, for example a coding region, genomic portion, etc.). Methods,
reagents, and reaction conditions for generating specific
polynucleotide sequences and multiplicities of polynucleotide
sequences are known in the art.
[0196] Generally, the polynucleotide is DNA, though, as noted
herein, the polynucleotide can comprise altered and/or modified
nucleotides, internucleotide linkages, ribonucleotides, etc. As
generally used herein, it is understood that "DNA" applies to
polynucleotide embodiments. In some aspects, namely the RNA
context, a canonical nucleotide includes a nucleotide comprising
the base uracil (U) (as well as respective forms such as
ribonucleoside, etc.).
[0197] Polynucleotides may be generated or isolated, e.g., from a
sample. Methods for preparing polynucleotide are well known in the
art. Methods for synthesizing polynucleotides, e.g., single and
double stranded DNA, from a template are well known in the art, and
include, for example, single primer isothermal amplification
(SPIA.TM.), Ribo-SPIA.TM., PCR, reverse transcription, primer
extension, limited primer extension, replication (including rolling
circle replication), strand displacement amplification (SDA), nick
translation, multiple displacement amplification (MDA). See, e.g.,
Kum, U.S. Pat. No.6,251,639 B1; Kurn, PCT Publication No. WO
02/00938; Kurn, U.S. Patent Publication No. 2003/0087251 A1;
Mullis, U.S. Pat. No. 4,582,877; Wallace, U.S. Pat. No. 6,027,923;
U.S. Pat. Nos. 5,508,178; 5,888,819; 6,004,744; 5,882,867;
5,710,028; 6,027,889; 6,004,745; 5,763,178; 5,011,769; see also
Sambrook (1989) "Molecular Cloning: A Laboratory Manual", second
edition; Ausebel (1987, and updates) "Current Protocols in
Molecular Biology"; Mullis, (1994) "PCR: The Polymerase Chain
Reaction". One or more methods known in the art can be used to
synthesize polynucleotides. Suitable methods include methods that
result in one single- or double-stranded polynucleotide comprising
a canonical nucleotide (for example, reverse transcription,
production of double stranded cDNA, a single round of DNA
replication), as well as methods that result in multiple single
stranded or double stranded copies or copies of the complement of a
template (for example, single primer isothermal amplification or
Ribo-SPIA.TM. or PCR). In one embodiment, a single-stranded
polynucleotide comprising a canonical nucleotide is synthesized
using single primer isothermal amplification. See Kurn, U.S. Pat.
No. 6,251,639 B1.
[0198] 1. Cleaving a Base Portion of a Canonical Nucleotide to
Create an Abasic Site
[0199] The polynucleotide comprising a canonical nucleotide is
treated with an agent, such as an enzyme, capable of generally,
specifically, or selectively cleaving a base portion of the
canonical deoxyribonucleoside to create an abasic site. As used
herein, "abasic site" encompasses any chemical structure remaining
following removal of a base portion (including the entire base) of
a canonical nucleotide with an agent capable of cleaving a base
portion of a nucleotide. In some embodiments, the agent (such as an
enzyme) catalyzes hydrolysis of the bond between the base portion
of the canonical nucleotide and a sugar in the canonical nucleotide
to generate an abasic site comprising a hemiacetal ring (in
some-embodiments, comprising an aldehyde moiety) and lacking the
base (interchangeably called "AP" site), though other cleavage
products are contemplated for use in the methods of the invention.
Suitable agents and reaction conditions for cleavage of base
portions of cleavable canonical nucleotides are known in the art,
and include the agents shown in Table 1.
1TABLE 1 Canonical nucleotide Agent Mechanism Reference dC
bisulfite dC + HSO.sub.3.sup.-.fwdarw. dU .fwdarw. AP site** U.S.
Pat. No. 6,017,704 dC Cytosine dC .fwdarw. dU .fwdarw. AP site**
Sohail et deaminase al, NAR 2003, 31: 2990-94. dG Acidic dG
.fwdarw. AP site conditions; alkylation (e.g., dimethyl sulfate
treatment) **UNG catalyzes dU .fwdarw. AP (abasic site).
[0200] One or more agents may be used. In some embodiments, the one
or more agents cleave a base portion of the same canonical
nucleotide. In other embodiments, the one or more agents cleave a
base portion of different canonical nucleotides. In some
embodiments, the agent is an enzyme. In other embodiments, the
agent is a chemical agent. In still other embodiments, the agent is
a reaction condition (such as presence of acidic conditions).
[0201] As is evident, in some embodiments dUTP is generated as an
intermediate and cleavage of a base portion of dUTP is necessary to
generate the abasic site. Methods for cleaving a base portion of
dUTP are known in the art. See, e.g., Lindahl, PNAS (1974)
71(9):3649-3653; Jendrisak, U.S. Pat. No. 6,190,865 B1; U.S. Pat.
No. 5,035,996; U.S. Pat. No. 5,418,149. For example, in some
embodiments, the enzyme cytosine deaminase is used to deaminate
dCTP, whereby dUTP is generated. UNG is then used to cleave the
base portion of dUTP, whereby an abasic site is generated. Thus, in
some embodiments, cytosine deaminase is used in conjunction with
UNG to generate an abasic site from the canonical nucleotide dCTP.
As used herein, "in conjunction" encompasses simultaneous treatment
(e.g., when cytosine deaminase and UNG cleavage occurs in the same
reaction mixture) and/or treatment at different times (e.g., when
cytosine deaminase and UNG treatment is conducted
sequentially).
[0202] As is evident, in some embodiments, different agents cleave
the base portion of the canonical nucleotide and cleave the
phosphodiester backbone at the abasic site. In other embodiments,
the agent that cleaves the base portion of the canonical nucleotide
is the same agent that cleaves a phosphodiester backbone at the
abasic site. In some embodiments, fragmentation and labeling is
performed under acidic conditions (in some embodiments, at pH 2-5;
in some embodiments, pH 3-3.5). Example 1 exemplifies reaction
conditions suitable for efficient labeling and fragmentation under
acidic conditions. In still other embodiments, ARP labeling and
fragmentation are performed under substantially similar reaction
conditions.
[0203] In some embodiments, cleavage of base portions of canonical
nucleotides is general, specific or selective cleavage (in the
sense that the agent (such as an enzyme) capable of cleaving a base
portion of a canonical nucleotide generally, specifically or
selectively cleaves the base portion of a particular canonical
nucleotide), whereby about 90%, about 85%, or about 80% of the base
portions cleaved are base portions of canonical nucleotides.
However, extent of cleavage can be less. Thus, reference to
specific cleavage is exemplary.
[0204] As noted herein, for convenience, cleavage of a base portion
of a canonical nucleotide (whereby an abasic site is generated) has
been described as a separate step. It is understood that this step
may be performed simultaneously with synthesis of the
polynucleotide comprising a canonical nucleotide (in embodiments
involving synthesis of the polynucleotide comprising the cleavable
canonical nucleotide), cleavage of the backbone at an abasic site
(fragmentation) and/or labeling at an abasic site. Example 1
exemplifies the use of acidic conditions for cleavage of a
canonical nucleotide (generally, dGTP) and cleavage of the
phosphodiester backbone at the abasic site.
[0205] In some embodiments involving synthesis of the
polynucleotide comprising the canonical nucleotide, the
polynucleotide comprising a canonical nucleotide is purified
following synthesis of the canonical polynucleotide (to eliminate,
for example, residual free canonical nucleotides that are present
in the reaction mixture). In other embodiments, there is no
intermediate purification between the synthesis of the
polynucleotide comprising the canonical nucleotide and subsequent
steps (such as cleavage of a base portion of the canonical
nucleotide and cleavage of a phosphodiester backbone at the abasic
site).
[0206] 2. Cleaving the Backbone at the Abasic Site of the
Polynucleotide Comprising an Abasic Site
[0207] Following generation of an abasic site, the backbone of the
polynucleotide is cleaved at the abasic site (i.e., the site
generated following cleavage of the base portion of the canonical
nucleotide) with an agent capable of effecting cleavage of the
backbone at the abasic site, as described herein.
[0208] 3. Labeling the Abasic Site and Detection
[0209] The abasic site is labeled, whereby a polynucleotide (or
polynucleotide fragment) comprising a label is generated as
described herein. In some embodiments, a polynucleotide fragment
comprising an abasic site is contacted with an agent capable of
labeling at the abasic site; whereby labeled fragments of the
polynucleotide are generated as described herein.
[0210] B. Methods for Labeling Nucleic Acids Comprising a Cleavable
Canonical Nucleotide
[0211] The invention provides methods for generating labeled
nucleic acid(s). The methods generally comprise cleavage of a base
portion of a canonical nucleotide present in a polynucleotide with
an agent capable of cleaving the base portion of the canonical
nucleotide; and labeling the abasic site, whereby labeled
polynucleotide(s) is generated. Generally, the polynucleotide
comprising a canonical nucleotide is labeled at the site of the
canonical nucleotide in the polynucleotide (following generation of
an abasic site by cleavage of a base portion of the canonical
nucleotide). The methods of the invention generate labeled
polynucleotide(s), which are useful for, for example, hybridization
to a microarray and other uses described herein.
[0212] The methods involve the following steps: (a) contacting the
polynucleotide comprising a canonical nucleotide with an agent
capable of cleaving a base portion of the canonical nucleotide,
whereby an abasic site is created; and (b) labeling the abasic site
in the polynucleotide comprising the abasic site, whereby labeled
polynucleotide(s) is generated.
[0213] For simplicity, individual steps of the labeling methods are
discussed below. It is understood, however, that the steps may be
performed simultaneously and in varied order, as discussed
herein.
[0214] In some embodiments, an basic site is generated at about
every 5, 10, 15, 20, 25, 30, 40, 50, 65, 75, 85, 100, 123, 150,
175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, 650 or more
nucleotides apart. In one embodiment, the abasic site is generated
about every 200 nucleotides, about every 100 nucleotide, or about
every 50 nucleotide. In another embodiment, the abasic site is
generated about every 50 to about 200 nucleotides.
[0215] The frequency (or spacing) of abasic sites in the resulting
polynucleotide comprising an abasic site (following cleavage of a
base portion of a canonical nucleotide, and thus the average size
of fragments generated using the methods of the invention (i.e.,
following cleavage of a phosphodiester backbone at a canonical
nucleotide), is controlled by variables known in the art,
including: frequency of canonical nucleotide(s) in the
polynucleotide (or other measures of nucleotide content of a
sequence, such as average G-C content), and the reaction conditions
used during generation of abasic site, as is further discussed
herein. The reaction conditions can be empirically determined, for
example, by assessing average fragment size generated using the
methods of the invention taught herein. In some embodiments, about
any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
more canonical bases are cleaved.
[0216] 1. Cleaving a Base Portion of a Canonical Nucleotide to
Create an Abasic Site
[0217] The polynucleotide comprising a canonical nucleotide is
treated with an agent, such as an enzyme, capable of generally,
specifically, or selectively cleaving a base portion of the
canonical deoxyribonucleoside to create an abasic site, as
described herein. 2. Labeling the Abasic Site and Detection
[0218] The abasic site is labeled, whereby a polynucleotide
comprising a label is generated, as described herein.
[0219] Methods for Preparing Polynucleotides (Or Fragments thereof)
and Immobilizationd on a Substrate
[0220] The invention provides methods for generating
polynucleotides or polynucleotide fragments and immobilization of
the fragments on a substrate (interchangeably termed a "surface",
herein). The methods generally comprise immobilizing the
polynucleotide, or fragments thereof, on a substrate, wherein the
polynucleotide or fragment thereof is immobilized at the abasic
site, wherein the polynucleotide, or fragment thereof, is generated
using any of the methods described herein. Optionally, the
polynucleotide comprising an abasic site can be labeled at an
abasic site according to the labeling methods described herein. The
methods of the invention generate polynucleotides, and fragments
thereof, immobilized on a substrate, for example, a microarray. In
some embodiments, one or more abasic site(s) are labeled (as
described herein) and one or more abasic site(s) are immobilized to
a substrate.
[0221] The methods involve the following steps: immobilizing a
polynucleotide (or polynucleotide fragments) on a substrate,
wherein the polynucleotide is immobilized to the substrate at the
abasic site; wherein the polynucleotide (or polynucleotide
fragment) comprising an abasic site is generated by any of the
methods described herein.
[0222] After generation of the polynucleotide comprising an abasic
site, the polynucleotide (or polynucleotide fragment, if the
backbone is cleaved) is immobilized to a substrate at the abasic
site. In embodiments involving cleavage of the backbone at an
abasic site (whereby fragments of the synthesized nucleic acid are
generated), the cleaved fragments are immobilized to a substrate at
the cleaved abasic site. Immobilizing a polynucleotide(s) is
useful, for example, to tag an analyte, or to create a microarray.
Single stranded polynucleotides (including polynucleotide
fragments) are particularly suitable for preparing microarrays
comprising the single stranded polynucleotides. Single stranded
polynucleotide fragments (in embodiments involving cleavage of the
phosphodiester backbone at an abasic site) are advantageous,
because the orientation of the fragment with respect to the
substrate (upon which the fragment is immobilized) can be
controlled by selection of the method used to cleave the
phosphodiester backbone, such that an abasic site is positioned at
the 3' end of a fragment or at the 5' end of a fragment.
Immobilizing polynucleotides in a defined orientation (e.g., at the
3' end, at the 5' end) enhances hybridization of complementary
oligonucleotides, and permits a higher density of
immobilization.
[0223] The polynucleotide comprising the abasic site is immobilized
to a substrate as follows: generally, reagents are used that are
capable of covalently or non-covalently attaching a reactive group
present in the abasic site to a reactive group present on a
substrate. For example, a common functional group exposed in an
abasic site (and therefore suitable for use in labeling) is the
aldehyde of the hemiacetal ring which can be covalently or
noncovalently attached to a reactive group on a suitable substrate
using reaction conditions that are known in the art. Suitable
sidechains (present on the substrate) to react with the aldehyde
(of the abasic site) include at least the following: substituted
hydrazines, hydrazides, or hydroxylamines (which readily form imine
bonds with aldehydes), and the related semicarbazide and
thiosemicarbazide groups, and other amines which can form stable
carbon-nitrogen double bonds, that can catalyze simultaneous
cleavage and binding (see Horn, Nucl. Acids. Res., (1988)
16:11559-71), or can be coupled to form stable conjugates, e.g. by
reductive amination.
[0224] The substrate to which the polynucleotide is to be
immobilized can be functionalized with suitable reactive groups
using methods known in the art. For example, a solid or semi-solid
substrate (e.g., silicon or glass slide) can be coated with
polymers (e.g., polyacrylamide, dextran, acrylamide, or latex)
comprising hydrazine, hydrazide, or amine derivatized substrates
(e.g. semicarbazides). Methods for functionalizing substrates with
suitable reactive groups are known in the art, and disclosed in,
for example, Luktanov, U.S. Pat. No. 6,339,147; Van Ness, U.S. Pat.
No. 5,667,976; Bangs Laboratories, Inc. TechNote 205 (available at
bangslabs.com); Ghosh, Anal. Biochem (1989) 178:43-51; O'Shannessy,
Anal. Biochem. (1990) 191:1-8; Wilchek, Methods Enzymol. (1987)
138:429-442; Baumgartner, Anal. Biochem. (1989) 181:182-189;
Zalipsky, Bioconjugate Chem. (1995) 6: 150-165, and references
cited therein.
[0225] Methods and reaction conditions for performing these
reactions are known in the art. See, e.g. Luktanov, U.S. Pat. No.
6,339,147; Van Ness, U.S. Pat. No. 5,667,976; Bangs Laboratories,
Inc. TechNote 205 (available at bangslabs.com); Ghosh, Anal.
Biochem (1989) 178:43-51; O'Shannessy, Anal. Biochem. (1990)
191:1-8; Wilchek, Methods Enzymol. (1987) 138:429-442; Baumgartner,
Anal. Biochem. (1989) 181:182-189; Zalipsky, Bioconjugate Chem.
(1995) 6: 150-165, and references cited therein. It is appreciated
that similar chemistry is described herein with respect to the
methods of labeling an abasic site (i.e., embodiments in which a
reactive group in the abasic site is covalently or non-covalently
attached to a suitable reactive group on a label). See, e.g.,
Srivastava, J. Biol. Chem. (1998) 273(33): 21203-209; Makrigiorgos,
Int J. Radiat. Biol. (1998) 74(1):99-109; Makrigiorgos, U.S. Pat.
No. 6,174,680 B1; Makrogiorgos, PCT Publication No. WO
00/39345.
[0226] In another example, the abasic site may be chemically
modified, then the modified abasic site covalently or
non-covalently attached to a suitable reactive group on a
substrate. For example, the aldehyde (in the abasic site) can be
oxidized or reduced (using methods known in the art), then
covalently immobilized to a substrate using, e.g., reductive
amination or various oxidative processes.
[0227] The substrate may consist of many materials, limited
primarily by capacity to immobilize (or, in some embodiments,
capacity for derivatization to immobilize) any of a number of
chemically reactive groups and compatibility with the synthetic
chemistry used to immobilize the polynucleotide comprising an
abasic site. The substrate can be a solid or semi-solid support,
which may be made, e.g., from glass, plastic (e.g., polystyrene,
polypropylene, nylon), polyacrylamide, nitrocellulose, or other
materials such as metals. As described herein, the substrate can be
functionalized, if necessary to add a suitable reactive group (to
which the abasic site is covalently or non-covalently immobilized).
The polynucleotides may also be spotted as a matrix on substrates
comprising paper, glass, plastic, polystyrene, polypropylene,
nylon, polyacrylamide, nitrocellulose, silicon, optical fiber or
any other suitable solid or semi-solid (e.g., thin layer of
polyacrylamide gel, assuming that the substrate is suitably
functionalized, as described herein (Khrapko, et al., DNA Sequence
(1991), 1:375-388)).
[0228] An array may be assembled as a two-dimensional matrix on a
planar substrate or may have a three-dimensional configuration
comprising pins, rods, fibers, tapes, threads, beads, particles,
microtiter wells, capillaries, cylinders and any other arrangement
suitable for hybridization and detection of template molecules. In
one embodiment the substrate to which the polynucleotide (or
fragments thereof) is immobilized is magnetic beads or particles.
In another embodiment, the solid substrate comprises an optical
fiber. In yet another embodiment, the polynucleotides are dispersed
in fluid phase within a capillary which, in turn, is immobilized
with respect to a solid phase.
[0229] In another embodiment, the substrate comprises a
polypeptide, a protein, a peptide, carbohydrates, cells,
microorganisms and fragments and products thereof, an organic
molecule, an inorganic molecule, carrier molecules, PEG,
amino-dextran, carbohydrates, supramolecular assemblies,
organelles, cells, microorganisms, organic molecules, inorganic
molecules, or any substance for which immobilization sites for
polynucleotides comprising abasic sites naturally exist, can be
created (e.g. by functionalizing or otherwise modifying the
substrate) or can be developed. In one embodiment, the substrate is
a polynucleotide.
[0230] The substrate may be an analyte. Typical analytes may
include, but are not limited to antibodies, proteins (including
enzymes), peptides, nucleic acid molecules or segments thereof,
carrier molecules, PEG, amino-dextran, carbohydrates,
supramolecular assemblies, organelles, cells, microorganisms,
organic molecules, inorganic molecules, or any substance for which
immobilization sites for polynucleotides comprising abasic sites
naturally exist, can be created (e.g. by functionalizing the
analyte) or can be developed.
[0231] It is understood that a substrate may be a member(s) of a
binding pair. Non-limiting examples of a binding pair include a
protein:protein binding pair, and a protein: antibody binding pair.
In another embodiment, polynucleotides (or fragments thereof) are
immobilized to (tag) a molecular library of substrates, e.g., a
molecular library of chemical compounds, a phage peptide display
library, or a library of antibodies.
[0232] In some embodiments, the substrate (to which the
polynucleotide is immobilized) is an enzyme, such that enhanced
detection of hybridization of the polynucleotide is provided. For
example, a polynucleotide immobilized to an enzyme can be
hybridized to a microarray, and hybridized polynucleotide detected
by contacting the microarray with a defined substrate.
[0233] In embodiments of the invention involving cleavage of the
phosphodiester backbone at an abasic site (whereby fragments of the
synthesized nucleic acid are generated), the cleaved fragments can
also be immobilized to a substrate using any method known in the
art for immobilization of a nucleic acid to a substrate. As used
herein, "immobilization" includes both covalent attachment and
non-covalent association. In one embodiment, a polynucleotide is
immobilized to a substrate directly via the abasic site. In another
embodiment, a polynucleotide is immobilized to a substrate directly
or indirectly via an attached or associated label.
[0234] For example, single or double stranded polynucleotide
fragments (generally single stranded) can be immobilized to a solid
or semi-solid support or substrate, which may be made, e.g., from
plastics, ceramics, metals, acrylamide, cellulose, nitrocellulose,
glass, polystyrene, polyethylene vinyl acetate, polypropylene,
polymethacrylate, polyethylene, polyethylene oxide, polysilicates,
polycarbonates, Teflon.RTM., fluorocarbons, nylon, silicon rubber,
polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans,
and polyamino acids, and other materials. Substrates may be
two-dimensional or three-dimensional in form, such as gels,
membranes, thin films, glasses, plates, cylinders, beads, magnetic
beads, optical fibers, woven fibers, microtiter well, capillaries,
etc. For example, the fragments can be contacted with a solid or
semi-solid substrate, such as a glass slide, which is coated with a
reactive group which will form a covalent link with the reactive
group that is on the polynucleotide fragment and become covalently
immobilized to the substrate.
[0235] Microarrays comprising the nucleotide fragments can be
fabricated using a Biodot (BioDot, Inc. Irvine, Calif.) spotting
apparatus and aldehyde-coated glass slides (CEL Associates,
Houston, Tex.). Polynucleotide fragments can be spotted onto the
aldehyde-coated slides following suitable functionalization, and
processed according to published procedures (Schena et al., Proc.
Natl. Acad. Sci. U.S.A. (1995) 93:10614-10619), provided suitable
care is taken to avoid interfering with other desired reactions at
the abasic sites. Arrays can also be printed by robotics onto
glass, nylon (Ramsay, G., Nature Biotechnol. (1998), 16:40-44),
polypropylene (Matson, et al., Anal Biochem. (1995), 224(1):110-6),
and silicone slides (Marshall, A. and Hodgson, J., Nature
Biotechnol. (1998), 16:27-31). Other approaches to array assembly
include fine micropipetting within electric fields (Marshall and
Hodgson, supra), and spotting the polynucleotides directly onto
positively coated plates. Methods such as those using amino propyl
silane surface chemistry are also known in the art, as disclosed at
http://www.cmt.corning.com and
http://cmgm.stanford.edu/pbrown/.
[0236] One method for making microarrays is by making high-density
polynucleotide arrays. Techniques are known for rapid deposition of
polynucleotides (Blanchard et al., Biosensors & Bioelectronics,
11:687-690). In principle, and as noted above, any type of array,
for example, dot blots on a nylon hybridization membrane, could be
used. However, as will be recognized by those skilled in the art,
very small arrays will frequently be preferred because
hybridization volumes will be smaller. Very small arrays may
include a high or low density of probes.
[0237] Methods for immobilizing polynucleotide fragments to
analytes (as described herein) are known in the art. See, e.g.,
U.S. Pat. Nos. 6,309,843; 6,306,365; 6,280,935; and 6,087,103 (and
methods discussed therein).
[0238] Applications Using the Labeling and/or Fragmentation and/or
Immobilization Methods of the Invention
[0239] The methods and compositions of the invention can be used
for a variety of purposes. For purposes of illustration, methods of
producing a hybridization probe or target, characterizing and/or
quantitating nucleic acid, detecting a mutation, preparing a
subtractive hybridization probe, detection (using the hybridization
probe), and determining a gene expression profile, using the
labeled and/or fragmented nucleic acids generated by the methods of
the invention, are described.
[0240] Immobilized polynucleotides, for example on a microarray,
prepared according to any of the methods of the invention, are also
useful for methods of analyzing and characterizing nucleic acids,
including methods of hybridizing nucleic acids, methods of
characterizing and/or quantitating nucleic acids, methods of
detecting a mutation in a nucleic acids, and methods of determining
a gene expression profile, as described below, and these
applications likewise apply to immobilized polynucleotides.
[0241] A. Method of Producing a Hybridization Probe or Target
[0242] The polynucleotides obtained by the methods of the invention
are useful as hybridization probes or targets. As used herein,
"probe" refers to a reference polynucleotide or oligonucleotide,
for example immobilized to a substrate or in solution, and "target"
refers to a polynucleotide or oligonucleotide from a sample to be
analyzed by hybridization to the probe. In some embodiments, the
target is detectably labeled. In other embodiments, a hybridization
target generated by a method of the invention is unlabeled and its
interaction with a probe is detected indirectly. Accordingly, in
one aspect, the invention provides methods for generating
hybridization targets, comprising generating labeled
polynucleotides using any of the methods described herein, and
using the labeled polynucleotides as a hybridization target. In
another embodiment, the invention provides methods for generating a
hybridization target, comprising generating labeled polynucleotide
fragments using any of the methods described herein, and using the
labeled polynucleotide fragments as a hybridization target. The
labeled polynucleotide (or polynucleotide fragments) can be
produced from any template, including RNA, DNA, genomic DNA
(including global genomic DNA amplification), and libraries
(including cDNA, genomic or subtractive hybridization library). The
invention also provides methods of hybridizing using the
hybridization targets to a probe as described herein. In another
aspect, the invention provides methods for generating hybridization
probes, comprising generating polynucleotides or polynucleotide
fragments using any of the methods described herein. In one
embodiment, a probe comprising a polynucleotide or polynucleotide
fragment generated by a method of the invention is immobilized to a
solid support. In one embodiment, the probe is labeled and is
immobilized to a support via the label (for example, indirect
immobilization via a member of a specific binding pair). In another
embodiment, the probe is unlabeled.
[0243] B. Characterization of Nucleic Acids
[0244] The labeled and/or fragmented nucleic acids obtained by the
methods of the invention are amenable to further
characterization.
[0245] The labeled and/or fragmented nucleic acids (i.e., products
of any of the methods described herein), can be analyzed using, for
example, probe hybridization techniques known in the art, such as
Southern and Northern blotting, and hybridizing to probe arrays.
They can also be analyzed by electrophoresis-based methods, such as
differential display and size characterization, which are known in
the art.
[0246] In one embodiment, the methods of the invention are utilized
to generate labeled and/or fragmented nucleic acids, and analyze
the labeled and/or fragmented nucleic acids by contact with a
probe. The labeled and/or fragmented nucleic acid can be produced
from any template known in the art, including RNA, DNA, genomic DNA
(including global genomic DNA amplification), and libraries
(including cDNA, genomic or subtractive hybridization library).
[0247] In one embodiment, the methods of the invention are utilized
to generate labeled and/or fragmented nucleic acids which are
analyzed (for example, detection and/or quantification) by
contacting them with, for example, microarrays (of any suitable
substrate, which includes glass, chips, plastic), beads, or
particles, that comprise suitable probes such as cDNA and/or
oligonucleotide probes. Thus, the invention provides methods to
characterize (for example, detect and/or quantify and/or identify)
a labeled and/or fragmented nucleic acid by analyzing the labeled
products, for example, by hybridization of the labeled products to,
for example, probes immobilized at, for example, specific locations
on a solid or semi-solid substrate, probes immobilized on defined
particles (including beads, such as Bead Array, Illumina), or
probes immobilized on blots (such as a membrane), for example
arrays, or arrays of arrays. Immobilized probes include immobilized
probes generated by the methods described herein, and also include
at least the following: cDNA and synthetic oligonucleotides, which
can be synthesized directly on the substrate.
[0248] Other methods of analyzing labeled products are known in the
art, such as, for example, by contacting them with a solution
comprising probes, followed by extraction of complexes comprising
the labeled products and probes from solution. The identity of the
probes provides characterization of the sequence identity of the
products, and thus by extrapolation can also provide
characterization of the identity of a template from which the
products were prepared (for example, the identity of an RNA in a
solution). For example, hybridization of the labeled products is
detectable, and the amount of specific labels that are detected is
proportional to the amount of the labeled products prepared from a
specific RNA sequence of interest. This measurement is useful for,
for example, measuring the relative amounts of the various RNA
species in a sample, which are related to the relative levels of
gene expression, as described herein. The amount of labeled
products (as indicated by, for example, detectable signal
associated with the label) hybridized at defined locations on an
array can be indicative of the detection and/or quantification of
the corresponding template RNA species in the sample.
[0249] Methods of characterization include sequencing by
hybridization (see, e.g., Dramanac, U.S. Pat. No. 6,270,961) and
global genomic hybridization (also termed comparative genome
hybridization) (see, e.g., Pinkel, U.S. Pat. No. 6,159,685).
[0250] In another aspect, the invention provides a method of
quantitating labeled and/or fragmented nucleic acids comprising use
of an oligonucleotide (probe) of defined sequence (which may be
immobilized, for example, on a microarray).
[0251] C. Mutation Detection Utilizing the Methods of the
Invention
[0252] The labeled and/or fragmented nucleic acids generated
according to the methods of the invention are also suitable for
analysis for the detection of any alteration in the template
nucleic acid sequence (from which the labeled and/or fragmented
nucleic acids are synthesized), as compared to a reference nucleic
acid sequence which is identical to the template nucleic acid
sequence other than the sequence alteration. The sequence
alterations may be sequence alterations present in the genomic
sequence or may be sequence alterations which are not reflected in
the genomic DNA sequences, for example, alterations due to post
transcriptional alterations, and/or mRNA processing, including
splice variants. Sequence alterations (interchangeably called
"mutations") include deletion, substitution, insertion and/or
transversion of one or more nucleotide.
[0253] Accordingly, the invention provides methods of detecting
presence or absence of a mutation in a template, comprising: (a)
generating a labeled polynucleotide, or fragments thereof, by any
of the methods described herein; and (b) analyzing the labeled
polynucleotide, or fragments thereof, whereby presence or absence
of a mutation is detected. In some embodiments, the labeled
polynucleotide, or fragments thereof, is compared to a labeled
reference template, or fragments thereof. Step (b) of analyzing the
labeled polynucleotide, or fragments thereof, whereby presence or
absence of a mutation is detected, can be performed by any method
known in the art. In some embodiments, probes for detecting
mutations are provided as a microarray.
[0254] Any alteration in the test nucleic acid sequence, such as
base substitution, insertions or deletion, could be detected using
this method. The method is expected to be useful for detection of
specific single base polymorphism, SNP, and the discovery of new
SNPs.
[0255] Other art recognized methods of analysis for the detection
of any alteration in the template nucleic acid sequence, as
compared to a reference nucleic acid sequence, are suitable for use
in the methods of the present invention. For example, essentially
any hybridization-based method of detection of mutations is
suitable for use with the labeled and/or fragmented nucleic acids
produced by the methods of the invention.
[0256] D. Methods of Preparing a Subtractive Hybridization
Probe
[0257] The labeled and/or fragmented nucleic acids methods of the
invention are particularly suitable for use in preparation of
labeled and/or fragmented subtractive hybridization probes. For
example, two nucleic acid populations, one sense and one antisense,
can be allowed to mix together with one population present in molar
excess ("driver"). Sequence present in both populations will form
hybrids, while sequences present in only one population remain
single-stranded. Thereafter, various well-known techniques are used
to separate the unhybridized molecules representing differentially
expressed sequences. See, e.g., Hamson et al., U.S. Pat. No.
5,589,339; Van Gelder, U.S. Pat. No. 6,291,170. Labeled and/or
fragmented subtractive hybridization probe is then labeled and/or
fragmented according to the methods of the invention described
herein.
[0258] E. Comparative Hybridization
[0259] In another aspect, the invention provides methods for
comparative hybridization (such as comparative genomic
hybridization), said method comprising: (a) preparing a first
population of labeled polynucleotides or fragments thereof from a
first template polynucleotide sample using any of the methods
described herein; (b) comparing hybridization of the first
population to at least one probe with hybridization of a second
population of labeled polynucleotides or fragments thereof. In some
embodiments, the at least one probe is a chromosomal spread. In
still other embodiments, the at least one probe is provided as a
microarray. In other embodiments, the second population of labeled
polynucleotides, or fragments thereof, are prepared from a second
polynucleotide sample using any of the methods described herein. In
some embodiments, the first population and second population
comprise detectably different labels. In some embodiments, step (b)
of comparing comprises determining amount of said products, whereby
the amount of the first and second polynucleotide templates is
quantified.
[0260] In some embodiments, comparative hybridization comprises
preparing a first population of labeled polynucleotides (which can
be polynucleotide fragments) according to any of the methods
described herein, wherein the template from which the first
population is synthesized is genomic DNA. A second population of
labeled polynucleotides (to which the first population is desired
to be compared) is prepared from a second genomic DNA template. The
first and second populations are labeled with different labels. The
hybridized first and second populations are mixed, and hybridized
to an array or chromosomal spread. The different labels are
detected and compared.
[0261] Reaction Conditions and Detection
[0262] Appropriate reaction media and conditions for carrying out
the methods of the invention are those that permit nucleic acid
synthesis according to the methods of the invention. Such media and
conditions are known to persons of skill in the art, and are
described in various publications, such as U.S. Pat. Nos.
6,190,865; 5,554,516; 5,716,785; 5,130,238; 5,194,370; 6,090,591;
5,409,818; 5,554,517; 5,169,766; 5,480,784; 5,399,491; 5,679,512;
PCT Publication No. WO 99/42618; Mol. Cell Probes (1992) 251-6; and
Anal. Biochem. (1993) 211:164-9. For example, a buffer may be Tris
buffer, although other buffers can also be used as long as the
buffer components are non-inhibitory to enzyme components of the
methods of the invention. The pH is from about 5 to about 11, more
preferably from about 6 to about 10, from about 7 to about 9, and
from about 7.5 to about 8.5. The reaction medium can also include
bivalent metal ions such as Mg.sup.2+ or Mn.sup.2+, at a final
concentration of free ions that is within the range of from about
0.01 to about 15 mM, and most preferably from about 1 to 10 mM. The
reaction medium can also include other salts, such as KCl or NaCl,
that contribute to the total ionic strength of the medium. For
example, the range of a salt such as KCl is preferably from about 0
to about 125 mM, more preferably from about 0 to about 100 mM, and
most preferably from about 0 to about 75 mM. The reaction medium
can further include additives that could affect performance of the
amplification reactions, but that are not integral to the activity
of the enzyme components of the methods. Such additives include
proteins such as BSA, single strand binding proteins (e.g., T4 gene
32 protein), and non-ionic detergents such as NP40 or Triton.
Reagents, such as DTT, that are capable of maintaining enzyme
activities can also be included. Such reagents are known in the
art. Where appropriate, an RNase inhibitor (such as Rnasin) that
does not inhibit the activity of the RNase employed in the method
(if any) can also be included. Any aspect of the methods of the
invention can occur at the same or varying temperatures. The
synthesis reactions (particularly, primer extension other than the
first and second strand cDNA synthesis steps, and strand
displacement) can be performed isothermally, which avoids the
cumbersome thermocycling process. The synthesis reaction is carried
out at a temperature that permits hybridization of oligonucleotides
(primer) to the template polynucleotide and primer extension
products, and that does not substantially inhibit the activity of
the enzymes employed. The temperature can be in the range of
preferably about 25.degree. C. to about 85.degree. C., more
preferably about 30.degree. C. to about 80.degree. C., and most
preferably about 37.degree. C. to about 75.degree. C. In some
embodiments that include RNA transcription, the temperature for the
transcription steps is lower than the temperature(s) for the
preceding steps. In these embodiments, the temperature of the
transcription steps can be in the range of preferably about
25.degree. C. to about 85.degree. C., more preferably about
30.degree. C. to about 75.degree. C., and most preferably about
37.degree. C. to about 70.degree. C.
[0263] Nucleotides that can be employed for synthesis of the
nucleic acids in the methods of the invention are provided in the
amount of from about 50 to about 2500 .mu.M, about 100 to about
2000 .mu.M, about 200 to about 1700 .mu.M, and about 250 to about
1500 .mu.M. The oligonucleotide components of the synthesis
reactions of the invention are generally in excess of the number of
template nucleic acid sequence to be replicated. They can be
provided at about or at least about any of the following: 10,
10.sup.2, 10, 10.sup.6, 10.sup.8, 10.sup.10, 10.sup.12 times the
amount oftarget nucleic acid. Composite primers can be provided at
about or at least about any of the following concentrations: 50 nM,
100 nM, 500 nM, 1000 nM, 2500 nM, 5000 nM.
[0264] Optionally, the polynucleotide template (i.e.,
polynucleotide comprising a canonical nucleotide, or polynucleotide
comprising a methylated nucleotide) can be treated with
hydroxylamine (or any other suitable agent) to remove any aldehydes
that may have formed spontaneously in the nucleic acid. See, e.g.,
Makrogiorgos, PCT Publication No. WO 00/39345.
[0265] For convenience, the cleavage of a base portion of that
polynucleotide by an enzyme capable of cleaving a base portion of
the canonical nucleotide (or capable of cleaving a base portion of
the methylated nucleotide, in embodiments involving methylation),
and the cleavage of the phosphodiester backbone at the abasic site,
are described as separate steps. It is understood that these steps
may be performed simultaneously.
[0266] Appropriate reaction media and conditions for carrying out
the cleavage of a base portion of a canonical nucleotide according
to the methods of the invention are those that permit cleavage of a
base portion of a canonical nucleotide. Such media and conditions
are known to persons of skill in the art, and are described in
various publications, such as Sohail et al, NAR 2003,31: 2990-94;
A. Sartori et al, JBC 2001, 276: 29979-29986; U.S. Pat. No.
6,017,704. In some embodiments involving cleavage of a canonical
nucleotide, dUTP is generated as an intermediate and cleavage of a
base portion of dUTP is necessary to generate the abasic site.
Methods for cleaving a base portion of dUTP are known in the art.
See, e.g., Lindahl, PNAS (1974) 71(9):3649-3653; Jendrisak, U.S.
Pat. No. 6,190,865 B1; U.S. Pat. No. 5,035,996; U.S. Pat. No.
5,418,149.
[0267] Appropriate reaction media and conditions for carrying out
the cleavage of a base portion of a methylated nucleotide according
to the methods of the invention are those that permit cleavage of a
base portion of a methylated nucleotide. Such media and conditions
are known to persons of skill in the art, and are described in
various publications, such as Wolffe et al., Proc. Nat. Acad. Sci.
USA 96:5894-5896, 1999); Zhu et al., Proc. Natl. Acad. Sci. USA
98:5031-6, 2001; Zhu et al., Nuc. Acid Res. 28:4157-4165, 2000;
Neddermann et al., J.B.C. 271:12767-74, 1996; Vairapandi &
Duker, Oncogene 13:933-938, 1996; Vairapandi et al., J. Cell.
Biochem. 79:249-260, 2000.
[0268] In embodiments involving cleavage of the phosphodiester
backbone, appropriate reaction media and conditions for carrying
out the cleavage of the phosphodiester backbone at an abasic site
according to the methods of the invention are those that permit
cleavage of the phosphodiester backbone at an abasic site. Such
media and conditions are known to persons of skill in the art, and
are described in various publications, such as Bioorgan. Med. Chem
(1991) 7:2351; Sugiyama, Chem. Res. Toxicol. (1994) 7: 673-83;
Horn, Nucl. Acids. Res., (1988) 16:11559-71); Lindahl, PNAS (1974)
71(9):3649-3653; Jendrisak, U.S. Pat. No. 6,190,865 B1; Shida,
Nucleic Acids Res. (1996) 24(22):4572-76; Srivastava, J. Biol Chem.
(1998) 273(13):21203-209; Carey, Biochem. (1999) 38:16553-60; Chem
Res Toxicol (1994) 7:673-683. For example, E. coli AP endonuclease
IV is added to reaction conditions as described above. AP
Endonuclease IV can be added at the same or different time as the
agent (such as an enzyme) capable of cleaving the base portion of a
canonical nucleotide.
[0269] In another example, nucleic acids containing abasic sites
are heated in a buffer solution containing an amine, for example,
25mM Tris-HCl and 1-5 mM magnesium ions, for 10-30 minutes at
70.degree. C. to 95.degree. C. Alternatively, 1.0 M piperidine (a
base) is added to polynucleotide comprising an abasic site which
has been precipitated with ethanol and vacuum dried. The solution
is then heated for 30 minutes at 90.degree. C. and lyophilized to
remove the piperidine. In another example, cleavage is affected by
treatment with basic solution, e.g., 0.2 M sodium hydroxide at
37.degree. for 15 minutes. See Nakamura (1998) Cancer Res.
58:222-225. In yet another example, incubation at 37 C with 100 mM
N,N'-dimethylethylenediamine acetate, pH 7.4 is used to cleave. See
McHugh and Knowland, (1995) Nucl. Acids Res. 23(10) 1664-1670; see
also co-pending co-owned U.S. patent application Ser. No.
10/441,663.
[0270] In one embodiment, the reaction conditions are the same for
the cleavage of a base portion of the canonical nucleotide (or in
embodiments, involving methylation analysis, cleavage of a base
portion of the methylated nucleotide) and for the cleavage of the
phosphodiester backbone at abasic sites. In another embodiment,
different reaction conditions are used for these reactions.
[0271] In embodiments involving labeling at an abasic site,
appropriate reaction media and conditions for carrying out the
labeling at an abasic site according to the methods of the
invention are those that permit labeling at an abasic site. Such
reaction mixtures and conditions are known to persons of skill in
the art, and are described in various publications, such as
co-pending co-owned U.S. patent application Ser. No. 10/441,663;
Makrigiorgos, PCT Publication No. WO 00/39345; Srivastava, J. Biol.
Chem. (1998) 273(33): 21203-209; Makrigiorgos, Int J. Radiat. Biol.
(1998) 74(1):99-109; Makrigiorgos, U.S. Pat. No. 6,174,680 B1;
Makrigiorgos, PCT Publication No. WO 00/39345; Boturyn (1999) Chem.
Res. Toxicol. 12:476-482. See, also, Adamczyk (1998) Bioorg. Med.
Chem. Lett. 8(24):3599-3602; Adamczyk (1999) Org. Lett.
1(5):779-781; Kow (2000) Methods 22(2):164-169; Molecular Probes
Handbook, Section 3.2 (www.probes.com); Horn (Nucl. Acids. Res.,
(1988) 16:11559-71). For example,
5-(((2-(carbohydrazino)-methyl)thio)acetyl)ami- nofluorescein,
aminooxyacetyl hydrazide (FARP); N-(aminooxyacetyl)-N'-(D-b-
iotinoyl) hydrazine, trifluorecetic acid salt (ARP); Alexa Fluor
555 (Molecular Probes); aminooxy-derivatized Alexa Fluor 555; and
other aldehyde-reactive reagents can be reacted with a
polynucleotide comprising abasic sites. The buffer can be sodium
citrate or sodium phosphate buffer, though other buffers are
acceptable as long as the buffer components are non-inhibitory to
enzyme components and/or desired chemical reactions used in the
methods of the invention. The pH is preferably from about 3 to
about 11, more preferably from about 4 to about 10, even more
preferably from about 4 to about 8, and most preferably from about
4 to about 7. The reaction can be conducted at room temperature to
85.degree. C. (in some embodiments, at about 55.degree. C.), though
other temperatures are suitable as long as the temperature is
non-inhibitory to enzyme components and/or desired chemical
reactions used in the methods of the invention. Generally, the
label (e.g. ARP or FARP) is added at about 1-10 mM, in some
embodiments at 2-5 mM, though other concentrations are suitable. If
an antibody label is used, conditions for antibody binding are
well-known in the art, and can be as described herein. Optionally,
a stop buffer can be used that neutralizes the pH of the labeling
reaction, thereby stopping the labeling reaction and optionally,
facilitating subsequent purification of labeled product.
[0272] In embodiments involving immobilization of a polynucleotide
at an abasic site, appropriate reaction media and conditions for
carrying out the immobilization at an abasic site according to the
methods of the invention are those that permit immobilization at an
abasic site. Such reaction mixtures and conditions are known to
persons of skill in the art, and are described in various
publications, such as Luktanov, U.S. Pat. No. 6,339,147; Van Ness,
U.S. Pat. No. 5,667,976; Bangs Laboratories, Inc. TechNote 205
(available at bangslabs.com); Ghosh, Anal. Biochem (1989)
178:43-51; O'Shannessy, Anal. Biochem. (1990) 191:1-8; Wilchek,
Methods Enzymol. (1987) 138:429-442; Baumgartner, Anal. Biochem.
(1989) 181:182-189; Zalipsky, Bioconjugate Chem. (1995) 6: 150-165,
and references cited therein. In some cases, the initial product
can be stabilized by reduction with sodium cyanoborohydride or
similar agents known in the art. See, e.g., O'Shannessy, supra.
[0273] In one embodiment, the foregoing components are added
simultaneously at the fragmentation and/or labeling and/or
immobilization processes. In another embodiment, components are
added in any order prior to or after appropriate timepoints during
the synthesis step. Such timepoints, some of which are noted below,
can be readily identified by a person of skill in the art. In these
embodiments, the reaction conditions and components may be varied
between the different reactions.
[0274] The fragmenting and/or labeling and/or immobilization
process can be stopped at various timepoints, and resumed at a
later time. Said timepoints can be readily identified by a person
of skill in the art. Methods for stopping the reactions are known
in the art, including, for example, cooling the reaction mixture to
a temperature that inhibits enzyme activity or heating the reaction
mixture to a temperature that destroys an enzyme. Methods for
resuming the reactions are also known in the art, including, for
example, raising the temperature of the reaction mixture to a
temperature that permits enzyme activity or replenishing a
destroyed (depleted) enzyme or other reagent. In some embodiments,
one or more of the components of the reactions is replenished prior
to, at, or following the resumption of the reactions.
Alternatively, the reaction can be allowed to proceed (i.e., from
start to finish) without interruption.
[0275] The reaction can be allowed to proceed without purification
of intermediate complexes, for example, to remove primer. Products
can be purified at various timepoints, which can be readily
identified by a person of skill in the art.
[0276] Compositions and Kits of the Invention
[0277] The invention also provides compositions and kits used in
the methods described herein. The compositions may be any
component(s), reaction mixture and/or intermediate described
herein, as well as any combination. For example, the invention
provides a composition comprising an agent (such as an enzyme)
capable of cleaving a base portion of a canonical nucleotide,
optionally an agent (such as an enzyme) capable of effecting
cleavage of a phosphodiester backbone at an abasic site, and an
agent capable of labeling an abasic site. In some embodiments, the
invention provides compositions further comprising a composite
primer, said composite comprising a DNA portion and a 5' RNA
portion. In still other embodiments, the 5' RNA portion of the
composite primer is adjacent to the 3' DNA portion, the RNA portion
of the composite primer consists of about 10 to about 20
nucleotides and the DNA portion of the composite primer consists of
about 7 to about 20 nucleotides. In still other embodiments, the
composition comprises a second, different composite primer. In some
embodiments, the RNA portion of the composite primer comprises the
following ribonucleotide sequence: 5'-GACGGAUGCGGUCU-3'. In still
other embodiments, the compositions further comprise one or more of
(a) a mixture of dATP, dTTP, dCTP, and dGTP; (b) a DNA polymerase;
and (c) RNAse H. In still other embodiments, the compositions
further comprise one or more of (a) MgCl2 solution; (b) acetic acid
solution; and optionally, (c) a stop buffer comprising 1.5M Tris,
pH 8.5. In some embodiments, the agent capable of cleaving the
phosphodiester backbone at an abasic site is an amine (such as
N,N'-dimethylethylenediamine); and/or E. coli Endonuclease IV. In
some embodiments, the agent capable of labeling an abasic site is
ARP, FARP, Alexa Fluor 555 hydrazide (Order No. A-20501, Molecule
Probes, Eugene Oreg.), and/or an aminooxy-modified Alexa Fluor 555
(see copending co-owned U.S. patent application Ser. No.
10/441,663).
[0278] In another example, the invention provides a composition
comprising an agent (such as an enzyme) capable of cleaving a base
portion of a methylated nucleotide, optionally, an agent (such as
an enzyme) capable of effecting cleavage of a phosphodiester
backbone at an abasic site, and an agent capable of labeling an
abasic site. In some embodiments, the agent capable of cleaving a
base portion of a methylated nucleotide is 5-methylcytosine DNA
glycosylase (5-MCDG), or 3-methyladenosine-DNA glycosylase. In some
embodiments, the agent capable of cleaving the phosphodiester
backbone at an abasic site is an amine (such as
N,N'-dimethylethylenediamine); and/or E. coli Endonuclease IV. In
some embodiments, the agent capable of labeling an abasic site is
ARP, FARP, Alexa Fluor 555 hydrazide (Order No. A-20501, Molecule
Probes, Eugene Oreg.), and/or an aminooxy-modified Alexa Fluor 555
(see copending co-owned U.S. patent application Ser. No.
10/441,663).
[0279] The compositions are generally in lyophilized or aqueous
form (if appropriate), preferably in a suitable buffer.
[0280] The invention also provides compositions comprising the
labeled and/or fragmented products described herein. Accordingly,
the invention provides a population of labeled and/or fragmented
polynucleotides, which are produced by any of the methods described
herein (or compositions comprising the products).
[0281] The invention also provides compositions comprising the
immobilized polynucleotides or immobilized polynucleotide fragments
described herein. In some embodiments, the immobilized
polynucleotide (or immobilized fragment, in embodiments involving
fragmentation) is labeled, as described herein. Accordingly, the
invention provides a population of immobilized polynucleotides or
immobilized polynucleotide fragments which are produced by any of
the methods described herein (or compositions comprising the
products).
[0282] The invention also provides reaction mixtures (or
compositions comprising reaction mixtures) which contain various
combinations of components described herein. Examples of reaction
mixtures have been described.
[0283] The invention provides kits for carrying out the methods of
the invention. Accordingly, a variety of kits are provided in
suitable packaging. The kits may be used for any one or more of the
uses described herein
[0284] The kits of the invention comprise one or more containers
comprising any combination of the components described herein, and
the following are examples of such kits.
[0285] In some embodiments, the invention provides kits comprising
an agent (such as an enzyme) capable of cleaving a base portion of
a canonical nucleotide, optionally an agent (such as an enzyme)
capable of effecting cleavage of a phosphodiester backbone at an
abasic site, and an agent capable of labeling an abasic site. In
some embodiments, the invention provides kits further comprising a
composite primer, said composite comprising a DNA portion and a 5'
RNA portion. In still other embodiments, the 5' RNA portion of the
composite primer is adjacent to the 3' DNA portion, the RNA portion
of the composite primer consists of about 10 to about 20
nucleotides and the DNA portion of the composite primer consists of
about 7 to about 20 nucleotides. In still other embodiments, the
kits comprise a second, different composite primer. In some
embodiments, the RNA portion of the composite primer comprises the
following ribonucleotide sequence: 5'-GACGGAUGCGGUCU-3'. In still
other embodiments, the kits further comprise one or more of (a) a
mixture of dATP, dTTP, dCTP, and dGTP; (b) a DNA polymerase; and
(c) RNAse H. In still other embodiments, the kits further comprise
one or more of (a) MgCl2 solution; (b) acetic acid solution; and
optionally, (c) a stop buffer comprising 1.5M Tris, pH 8.5. In some
embodiments, the invention provides a kit comprising RNAseH, and an
agent (such as an enzyme) capable of cleaving a base portion of a
canonical nucleotide. RNase H. In some embodiments, the invention
provides a kit comprising a composite primer comprising an RNA
portion and a 3' DNA portion and instructions for any of the
methods for labeling and/or fragmenting a polynucleotide comprising
a canonical nucleotide described herein. In some embodiments, the
agent capable of cleaving the phosphodiester backbone at an abasic
site is an amine (such as N,N'-dimethylethylenediamine); and/or E.
coli Endonuclease IV. In some embodiments, the agent capable of
labeling an abasic site is ARP, FARP, Alexa Fluor 555 hydrazide
(Order No. A-20501, Molecule Probes, Eugene Oreg.), and/or an
aminooxy-modified Alexa Fluor 555 (see copending co-owned U.S.
patent application Ser. No. 10/441,663).
[0286] In another example, the invention provides kits comprising
an agent (such as an enzyme) capable of cleaving a base portion of
a methlylated nucleotide, optionally, an agent (such as an enzyme)
capable of effecting cleavage of a phosphodiester backbone at an
abasic site, and an agent capable of labeling an abasic site. In
some embodiments, the agent capable of cleaving a base portion of a
methylated nucleotide is 5-methylcytosine DNA glycosylase (5-MCDG),
or 3-methyladenosine-DNA glycosylase. In some embodiments, the
agent capable of cleaving the phosphodiester backbone at an abasic
site is an amine (such as N,N'-dimethylethylenediamine); and/or E.
coli Endonuclease IV. In some embodiments, the agent capable of
labeling an abasic site is ARP, FARP, Alexa Fluor 555 hydrazide
(Order No. A-20501, Molecule Probes, Eugene Oreg.), and/or an
aminooxy-modified Alexa Fluor 555 (see copending co-owned U.S.
patent application Ser. No. 10/441,663).
[0287] Kits may also include one or more suitable buffers (as
described herein). One or more reagents in the kit can be provided
as a dry powder, usually lyophilized, including excipients, which
on dissolution will provide for a reagent solution having the
appropriate concentrations for performing any of the methods
described herein. Each component can be packaged in separate
containers or some components can be combined in one container
where cross-reactivity and shelf life permit.
[0288] The kits of the invention may optionally include a set of
instructions, generally written instructions, although electronic
storage media (e.g., magnetic diskette or optical disk) containing
instructions are also acceptable, relating to the use of components
of the methods of the invention for the intended methods of the
invention, and/or, as appropriate, for using the products for
purposes such as, for example preparing a hybridization probe or
target, expression profiling, preparing a microarray, or
characterizing a nucleic acid. The instructions included with the
kit generally include information as to reagents (whether included
or not in the kit) necessary for practicing the methods of the
invention, instructions on how to use the kit, and/or appropriate
reaction conditions.
[0289] The component(s) of the kit may be packaged in any
convenient, appropriate packaging. The components may be packaged
separately, or in one or multiple combinations.
[0290] The relative amounts of the various components in the kits
can be varied widely to provide for concentrations of the reagents
that substantially optimize the reactions that need to occur to
practice the methods disclosed herein and/or to further optimize
the sensitivity of any assay.
[0291] The following examples are provided to illustrate, but not
to limit, the invention.
EXAMPLES
Example 1
Acid-Catalyzed Fragmentation and Labeling of cDNA
[0292] Single stranded cDNA was prepared from universal human total
RNA (Stratagene, La Jolla Calif.). Pooled purified cDNA product at
a concentration of 145 .mu.g/mL in water was aliquoted into each of
five 200 .mu.L PCR tubes, dispensing 13 .mu.L or 1.89 .mu.g into
each tube. 12 .mu.L water was then added to each tube, followed by
2.5 .mu.L of 0.5 M glycolic acid buffer (prepared by dissolving
glycolic acid in water at a concentration of 1.0 M, adjusting pH
with 1 M NaOH, then diluting to 0.5 M with water). Three tubes
received buffer at a pH of 3.0, and two received buffer of pH 3.5.
The pH 3 tubes were heated at 95.degree. C. for 5 minutes and
65.degree. for 5 minutes or 30 minutes. The pH 3.5 tubes were
heated at 65.degree. C. for 5 minutes or 30 minutes, all in a MJ
Research Peltier Effect thermal cycler. Tubes were then held
briefly on ice for the next step.
[0293] To each tube was added 5 .mu.L of 0.5 M acetate buffer pH
4.33 (prepared by pH adjustment of acetic acid with NaOH), followed
by 1 .mu.L of 1 M NaOH in each pH 3.0 tube and 0.7 .mu.L of 1 M
NaOH in pH 3.5 tubes. All tubes then received 2 .mu.L of 0.2 M
MgCl.sub.2 and 2.7 .mu.L of a biotinylating reagent. This reagent
contained 11.7 mg/mL of ARP (N-(aminooxyacetyl)-N'-(D-biotinoyl)
hydrazine, trifluoroacetic acid salt) (Molecular Probes, Eugene,
Oreg.) dissolved in a 22.5 mM solution of dibasic sodium phosphate.
All tubes were then incubated at 50.degree. C. for 30 minutes, then
10 .mu.L of 1 M Tris, pH 8.5 was added to each tube.
[0294] The contents of each tube were purified using a single
CentriSep size exclusion column (Princeton Separations, Adelphia,
N.J.) following the manufacturer's instructions. Concentration of
recovered product was estimated from A.sub.260 assuming that 33
.mu.g/mL of DNA gives A.sub.260=1.0. It was noted that the
A.sub.260/A.sub.280 ratio for the product heated at 95.degree. C.
was 1.75, while all other products gave ratios >1.9. Recoveries
were 60-69%.
[0295] Extent of fragmentation and labeling was determined using a
gel shift assay. Two aliquots of product, each containing 50 ng DNA
(3.1-3.5 .mu.L), were mixed in PCR tubes with 0.5 .mu.L of
10.times. TE buffer (0.1 M Tris, 10 mM EDTA, pH 7.5). To one
aliquot was added 3 .mu.L of a 2.5 mg/mL solution of streptavidin
(Sigma-Aldrich, St. Louis, Mo.) in water. After about 3 minutes
incubation, 3 .mu.L of 30% glycerol/bromophenol blue was added to
each tube, and products were loaded and run on Novex 4-20% TBE gels
(Invitrogen, Carlsbad, Calif.) following manufacturer's
instructions. Bands were visualized by staining with Sybr Green II
(Molecular Probes, Eugene, Oreg.) diluted 1:5,000 in gel buffer for
6 minutes, followed by imaging with an AlphaImager. Unreacted cDNA
product and molecular weight markers were included as controls.
[0296] An image of the gel is shown in FIG. 1. Lane 1 is molecular
weight markers, lane 2 is unreacted starting material, and
subsequent lanes are paired products without or with streptavidin
treatment. Lane 3 (5 min at 95.degree. C., pH 3.0) shows extensive
fragmentation of product compared to control in Lane 2. Average
apparent size appears to be reduced to 400-500 bases, and the high
molecular weight material in Lane 2 is virtually absent. Lane 4
shows that nearly all of the fragmented DNA is retarded by
streptavidin, appearing as a high molecular weight smear, much of
which does not enter the gel. The other extreme was represented by
lanes 11 and 12 (5 min at 65.degree. C., pH 3.5), where the length
without streptavidin appeared identical to control, and some but
not all of the product was shifted to higher molecular weight by
streptavidin. The other pairs of lanes represented intermediate
conditions in which little fragmentation was evident, but various
extents of labeling were seen, as measured by streptavidin shift.
Compared to lanes 11 and 12, longer time (30 min in Lanes 9 and
10), lower pH (pH 3.0 in Lanes 5 and 6), or both lower pH and
longer time (30 min at pH 3.0 in Lanes 7 and 8) resulted in
increased labeling.
[0297] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entireties for all
purposes.
* * * * *
References