U.S. patent application number 12/543450 was filed with the patent office on 2010-02-18 for dna methylation detection methods.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Shoulian Dong, Junko F. Stevens.
Application Number | 20100041057 12/543450 |
Document ID | / |
Family ID | 41681504 |
Filed Date | 2010-02-18 |
United States Patent
Application |
20100041057 |
Kind Code |
A1 |
Dong; Shoulian ; et
al. |
February 18, 2010 |
DNA METHYLATION DETECTION METHODS
Abstract
The present teachings provide DNA methylation quantification
methods that avoid bisulfite treatment of DNA. Methylation-specific
binding proteins (MeDNA binding proteins) and non-methylation
specific binding proteins (non-MeDNA binding proteins) are employed
in various embodiments to modulate the accessibility of nucleic
acids to primer extension reactions. After selectively removing the
target nucleic acids, the extension products can be analyzed and
methylation quantitated. In some embodiments, the analysis
comprises real-time PCR.
Inventors: |
Dong; Shoulian; (San Jose,
CA) ; Stevens; Junko F.; (Menlo Park, CA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
41681504 |
Appl. No.: |
12/543450 |
Filed: |
August 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61089856 |
Aug 18, 2008 |
|
|
|
Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6827 20130101; C12Q 2537/164 20130101; C12Q 2522/101
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of quantitating methylation in a target nucleic acid
comprising; treating a target nucleic acid with a MeDNA binding
protein, wherein the MeDNA binding protein forms a blocking complex
with a methylated cytosine in the target nucleic acid, wherein the
methylated cytosine in the target nucleic acid is near a first
target specific primer binding site; extending a first target
specific primer hybridized to the first target specific primer
binding site to form a target nucleic acid extension product;
degrading the target nucleic acid; amplifying the target nucleic
acid extension product; determining the difference between the
amount of the target nucleic acid with the amount of a control
nucleic acid lacking a methylated cytosine; and, quantitating
methylation in the target nucleic acid.
2. The method according to claim 1 wherein the amplifying comprises
a polymerase chain reaction comprising a first target specific
primer and a second target specific primer.
3. The method according to claim 1 wherein the degrading comprises
treating the target nucleic acid with a nuclease, wherein the
target nucleic acid extension product is resistant to the nuclease
due to a blocking moiety in the first target specific primer.
4. A method of quantitating methylation in a target nucleic acid
comprising; treating, in any order, (a) the target nucleic acid
with a MeDNA binding protein, wherein the MeDNA binding protein
forms a blocking complex with a methylated cytosine in the target
nucleic acid, wherein the methylated cytosine is disposed between a
first target specific primer binding site and a second target
specific primer binding site; and, (b) a control nucleic acid with
a MeDNA binding protein, wherein the MeDNA binding protein fails to
form a blocking complex with an unmethylated cytosine in the
control nucleic acid, wherein the non-methylated cytosine is
disposed between a first control specific primer binding site and a
second control specific primer binding site; extending, in any
order, (a) a first target specific primer hybridized to the first
target specific primer binding site to form a target nucleic acid
extension product; and, (b) a first control specific primer
hybridized to the first control specific primer binding site to
form a control nucleic acid extension product; degrading, in any
order, (a) the target nucleic acid; and, (b) the control nucleic
acid; amplifying, in any order, (a) the target nucleic acid
extension product in a polymerase chain reaction comprising a first
target specific primer and a second target specific primer; and,
(b) the control nucleic acid extension product in a polymerase
chain reaction comprising a first control specific primer and a
second control specific primer; determining the difference between
the amount of target nucleic acid with the amount of control
nucleic acid; and, quantitating methylation in the target nucleic
acid.
5. The method of claim 4 wherein the determining comprises;
measuring, in any order, (a) a first Ct value associated with the
amount of the target nucleic acid, and, (b) a second Ct value
associated with the amount of the control nucleic acid; and,
quantitating methylation in the target nucleic by comparing the
first Ct value with the second Ct value.
6. The method of claim 4 wherein the first Ct value is higher than
the second Ct value, and the target nucleic acid is more methylated
than the control nucleic acid.
7. The method of claim 4 wherein the first Ct value is lower than
the second Ct value, and the target nucleic acid is less methylated
than the control nucleic acid.
8. The method of claim 4 wherein the target nucleic acid and the
control nucleic acid comprise the same first primer binding site
and the same second primer binding site.
9. The method according to claim 4 wherein the target nucleic acid
is amplified in a separate reaction vessel from the control nucleic
acid.
10. The method of claim 9 wherein the target nucleic acid and the
control nucleic acid comprise the same first primer binding site
and the same second primer binding site and are amplified with a
common first primer and a common second primer.
11. The method according to claim 4 wherein the target nucleic acid
is amplified in a same reaction vessel as the control nucleic
acid.
12. The method of claim 11 wherein the target nucleic acid and the
control nucleic acid comprise a different first primer binding site
and a different second primer binding site and are amplified with a
different first primer and a different second primer.
13. The method according to claim 4 wherein the quantitating
comprises measuring an interchelating dye.
14. The method according to claim 4 wherein the determining
comprises; measuring displacement of a target sequence specific
probe, wherein the target sequence specific probe hybridizes to a
region of the target nucleic acid extension product, or complement
to the target nucleic acid extension product, disposed between the
first target specific primer binding site and the second target
specific primer binding site; measuring displacement of a control
sequence specific probe, wherein the control sequence specific
probe hybridizes to a region of the control nucleic acid extension
product, or complement to the control nucleic acid extension
product, disposed between the first control specific primer binding
site and the second control specific primer binding site.
15. The method according to claim 4, wherein the treating with the
MeDNA binding protein further comprises a cofactor.
16. The method according to claim 15 wherein the cofactor is
selected from the group consisting of S-adenosylmethionine,
S-adenosylhomocysteine and sinefungin. In a preferred embodiment,
S-adenosylhomocycteine is used.
17. The method according to claim 4 wherein the first primer
hybridizes upstream from the blocking complex formed by the MeDNA
binding protein and the methylated cytosine in the target nucleic
acid, and further extension of the first primer is blocked.
18. The method according to claim 4 wherein the first primer
hybridizes on the methylated cytosine on the blocking complex
formed by the MeDNA binding protein and the methylated cytosine in
the target nucleic acid, and initial extension of the first primer
is blocked.
19. The method according to claim 4 wherein the first primer does
not hybridize to the methylated cytosine on the blocking complex
formed by the MeDNA binding protein and the methylated cytosine in
the target nucleic acid.
20. A kit for quantitating methylation in a target nucleic acid
comprising; a Me-DNA binding protein; a first target specific
primer; a second target specific primer; a first control specific
primer; a second control specific primer; and, a polymerase.
21. The kit according to claim 20 wherein; the first control
specific primer is a different sequence from the first target
specific primer; and, the second control specific primer is a
different sequence from the second target specific primer.
22. The kit according to claim 20 wherein; the first control
specific primer is a same sequence as the first target specific
primer; and, the second control specific primer is a same sequence
as the second target specific primer.
23. The kit according to claim 20 further comprising a control
sequence specific probe.
24. The kit according to claim 23 further comprising a target
sequence specific probe, wherein the control sequence specific
probe is a different sequence from the target sequence specific
probe.
25. The kit according to claim 23 further comprising a target
sequence specific probe, wherein the control sequence specific
probe is a same sequence as the target sequence specific probe.
26. The kit according to claim 20 further comprising a
cofactor.
27. The kit according to claim 26 wherein the cofactor is selected
from the group consisting of S-adenosylmethionine,
S-adenosylhomocysteine and sinefungin.
28. A method of quantitating methylation in a target nucleic acid
comprising; treating a control nucleic acid with a non-MeDNA
binding protein, wherein the non-MeDNA binding protein forms a
blocking complex with an unmethylated cytosine in the control
nucleic acid, wherein the unmethylated cytosine in the control
nucleic acid is near a first control specific primer binding site;
extending a first primer hybridized to the first primer binding
site to form a control nucleic acid extension product; degrading
the control nucleic acid; amplifying the control nucleic acid
extension product in a polymerase chain reaction comprising a first
control specific primer and a second control specific primer;
determining the difference between the amount of the control
nucleic acid with the amount of a target nucleic acid containing a
methylated cytosine; and, quantitating methylation in the target
nucleic acid.
29. The method according to claim 28 wherein the amplifying
comprises a polymerase chain reaction comprising a first target
specific primer and a second target specific primer.
30. The method according to claim 28 wherein the degrading
comprises treating the target nucleic acid with a nuclease, wherein
the target nucleic acid extension product is resistant to the
nuclease due to a blocking moiety in the first target specific
primer.
31. A method of quantitating methylation in a target nucleic acid
comprising; treating, in any order, (a) the control nucleic acid
with a non-MeDNA binding protein, wherein the non-MeDNA binding
protein forms a blocking complex with an unmethylated cytosine in
the control nucleic acid lacking, wherein the unmethylated cytosine
is disposed between a first control-specific primer binding site
and a second control-specific primer binding site; and, (b) a
target nucleic acid with a non-MeDNA binding protein, wherein the
non-MeDNA binding protein fails to form a blocking complex with a a
methylated cytosine in the target nucleic acid, wherein the
methylated cytosine is disposed between a first target specific
primer binding site and a second target specific primer binding
site; extending, in any order, (a) a first target specific primer
hybridized to the first target specific primer binding site to form
a target nucleic acid extension product; and, (b) a first control
specific primer hybridized to the first control specific primer
binding site to form a control nucleic acid extension product;
degrading, in any order, (a) the target nucleic acid; and, (b) the
control nucleic acid; amplifying, in any order, (a) the target
nucleic acid extension product in a polymerase chain reaction
comprising a first target specific primer and a second target
specific primer; and, (b) the control nucleic acid extension
product in a polymerase chain reaction comprising a first control
specific primer and a second control specific primer; determining
the difference between the amount of target nucleic acid with the
amount of control nucleic acid; and, quantitating methylation in
the target nucleic acid.
32. The method of claim 31 wherein the determining comprises;
measuring, in any order, (a) a first Ct value associated with the
amount of the target nucleic acid, and, (b) a second Ct value
associated with the amount of the control nucleic acid; and,
quantitating methylation in the target nucleic by comparing the
first Ct value with the second Ct value.
33. The method of claim 31 wherein the first Ct value is higher
than the second Ct value, and the target nucleic acid is less
methylated than the control nucleic acid.
34. The method of claim 31 wherein the first Ct value is lower than
the second Ct value, and the target nucleic acid is more methylated
than the control nucleic acid.
35. The method of claim 31 wherein the target nucleic acid and the
control nucleic acid comprise the same first primer binding site
and the same second primer binding site.
36. The method according to claim 31 wherein the target nucleic
acid is amplified in a separate reaction vessel from the control
nucleic acid.
37. The method of claim 36 wherein the target nucleic acid and the
control nucleic acid comprise the same first primer binding site
and the same second primer binding site and are amplified with a
common first primer and a common second primer.
38. The method according to claim 31 wherein the target nucleic
acid is amplified in a same reaction vessel as the control nucleic
acid.
39. The method of claim 38 wherein the target nucleic acid and the
control nucleic acid comprise a different first primer binding site
and a different second primer binding site and are amplified with a
different first primer and a different second primer.
40. The method according to claim 31 wherein the quantitating
comprises measuring an interchelating dye.
41. The method according to claim 31 wherein the determining
comprises; measuring displacement of a target sequence specific
probe, wherein the target sequence specific probe hybridizes to a
region of the target nucleic acid extension product, or complement
to the target nucleic acid extension product, disposed between the
first target specific primer binding site and the second target
specific primer binding site; measuring displacement of a control
sequence specific probe, wherein the control sequence specific
probe hybridizes to a region of the control nucleic acid extension
product, or complement to the control nucleic acid extension
product, disposed between the first control specific primer binding
site and the second control specific primer binding site.
42. The method according to claim 31, wherein the treating with the
non-MeDNA binding protein further comprises a cofactor.
43. The method according to claim 42 wherein the cofactor is
SELECTED FROM THE GROUP CONSISTING OF S-adenosylmethionine,
S-adenosylhomocysteine and sinefungin. In a preferred embodiment,
S-adenosylhomocycteine is used.
44. The method according to claim 31 wherein the first primer
hybridizes upstream from the blocking complex formed by the
non-MeDNA binding protein and the unmethylated cytosine in the
control nucleic acid, and further extension of the first primer is
blocked.
45. The method according to claim 31 wherein the first primer
hybridizes on the unmethylated cytosine on the blocking complex
formed by the non-MeDNA binding protein and the unmethylated
cytosine in the control nucleic acid, and initial extension of the
first primer is blocked.
46. The method according to claim 31 wherein the first primer does
not hybridize to the unmethylated cytosine on the blocking complex
formed by the non-MeDNA binding protein and the unmethylated
cytosine in the target nucleic acid.
47. A kit for quantitating methylation in a target nucleic acid
comprising; a non-MeDNA binding protein; a first target specific
primer; a second target specific primer; a first control specific
primer; a second control specific primer; and, a polymerase.
48. The kit according to claim 47 wherein; the first control
specific primer is a different sequence from the first target
specific primer; and, the second control specific primer is a
different sequence from the second target specific primer.
49. The kit according to claim 47 wherein; the first control
specific primer is a same sequence as the first target specific
primer; and, the second control specific primer is a same sequence
as the second target specific primer.
50. The kit according to claim 47 further comprising a control
sequence specific probe.
51. The kit according to claim 50 further comprising a target
sequence specific probe, wherein the control sequence specific
probe is a different sequence from the target sequence specific
probe.
52. The kit according to claim 50 further comprising a target
sequence specific probe, wherein the control sequence specific
probe is a same sequence as the target sequence specific probe.
53. The kit according to claim 47 further comprising a
cofactor.
54. The kit according to claim 53 wherein the cofactor is selected
from the group consisting of S-adenosylmethionine,
S-adenosylhomocysteine and sinefungin.
Description
FIELD
[0001] The present teachings pertain to methods and kits for
quantitating cytosine methylation in target nucleic acids.
INTRODUCTION
[0002] Epigenomic changes to DNA provide another channel of
information on which natural selection can act (see Goldberg et
al., Cell, 128: 635-638). Increasing attention is being paid to
methylation of bases in nucleic acids as one important epigenomic
change. Methylation of bases can take different forms. For example,
methylation of DNA by the DNA adenine methyltransferase (Dam)
provides an epigenetic signal that influences and regulates
numerous physiological processes in the bacterial cell including
chromosome replication, mismatch repair, transposition, and
transcription (see Heusipp et al., Int J Med Microbiol. 2007
February; 297(1):1-7. Epub 2006 Nov. 27 for a review). Also,
methylation of cytosine in mammals at CpG dinucleotides correlates
with transcriptional repression, and plays a crucial role in gene
regulation and chromatin organization during embryogenesis and
gametogenesis (Goll and Bestor (2006) Annu. Rev. Biochem. 74,
481-514).
[0003] One method of measuring the presence of cytosine methylation
takes advantage of the ability of the converting agent bisulfite to
convert non-methylated cytosines to uracil (See Boyd et al., Anal
Biochem. 2004 Mar. 15; 326(2):278-80, Anal Biochem. 2006 Jul. 15;
354(2):266-73. Epub 2006 May 6, and Nucleosides Nucleotides Nucleic
Acids. 2007; 26(6-7):629-34. After such conversion, a sequence
amplified in a PCR bears thymine at those residues that were
originally unmethylated cytosine. However, methylated cytosines are
protected from such bisulfite treatment. Accordingly, the presence
of a thymine at a location known to normally contain cytosine
reflects that the original cytosine was unmethylated. Conversely,
the presence of a cytosine at a location known to normally contain
cytosine reflects that the original cytosine was methylated.
[0004] Following bisulfite conversion, and PCR amplification,
sequences containing a large number of unmethylated cytosines will
have a low complexity, since the non-methylated cytosines will have
been converted to thymine, and the resulting sequence will be
dominated by only three bases (A, G, and T). Such low complexity
sequences can be difficult to map to a region (locus) of the
genome. That is, when a low complexity nucleic acid is sequenced,
it can be difficult to know what part of the genome the sequence
comes from. Such a problem is particularly acute in various
sequencing approaches that employ short read-lengths.
[0005] Bisulfite treatment is also problematic because of limited
sample size. Treatment is harsh, and small amounts of starting
material are not easily analyzed using bisulfite.
SUMMARY
[0006] A method of quantitating methylation in a target nucleic
acid comprising;
[0007] treating a target nucleic acid with a MeDNA binding protein,
wherein the MeDNA binding protein forms a blocking complex with a
methylated cytosine in the target nucleic acid, wherein the
methylated cytosine in the target nucleic acid is near a first
target specific primer binding site;
[0008] extending a first target specific primer hybridized to the
first target specific primer binding site to form a target nucleic
acid extension product;
[0009] degrading the target nucleic acid;
[0010] amplifying the target nucleic acid extension product;
[0011] determining the difference between the amount of the target
nucleic acid with the amount of a control nucleic acid lacking a
methylated cytosine; and, quantitating methylation in the target
nucleic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows one illustrative embodiment according to the
present teachings.
[0013] FIG. 2 shows one illustrative embodiment according to the
present teachings.
[0014] FIG. 3 shows one illustrative embodiment according to the
present teachings.
[0015] FIG. 4 shows one illustrative embodiment according to the
present teachings.
[0016] FIG. 5 shows one illustrative embodiment according to the
present teachings.
[0017] FIG. 6 shows one illustrative embodiment according to the
present teachings.
[0018] FIG. 7 shows one example according to the present
teachings.
[0019] FIG. 8 shows effect of antibody by amount on Ct and delta
Ct.
[0020] FIG. 9 shows effect of antibody amount/duplex reaction
[0021] FIG. 10 shows effect of antibody amount/duplex reaction:
reduced Ct shift of 0 Me by increasing RT amount
[0022] FIG. 11 shows effect of antibody amount/duplex reaction with
normalized template/high salt
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0023] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not intended to limit the scope of the
current teachings. In this application, the use of the singular
includes the plural unless specifically stated otherwise. Also, the
use of "comprise", "contain", and "include", or modifications of
those root words, for example but not limited to, "comprises",
"contained", and "including", are not intended to be limiting. The
use of "or" means "and/or" unless stated otherwise. The term and/or
means that the terms before and after can be taken together or
separately. For illustration purposes, but not as a limitation, "X
and/or Y" can mean "X" or "Y" or "X and Y".
[0024] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the described
subject matter in any way. All literature and similar materials
cited in this application, including, patents, patent applications,
articles, books, treatises, and internet web pages are expressly
incorporated by reference in their entirety for any purpose. In the
event that one or more of the incorporated literature and similar
defines or uses a term in such a way that it contradicts that
term's definition in this application, this application controls.
While the present teachings are described in conjunction with
various embodiments, it is not intended that the present teachings
be limited to such embodiments. On the contrary, the present
teachings encompass various alternatives, modifications, and
equivalents, as will be appreciated by those of skill in the
art.
SOME DEFINITIONS
[0025] As used herein, the term "degrading" refers to removal of
unwanted nucleic acids in a reaction. Such degradation can be
achieved, for example, by employing first primers in the primer
extension reaction that contain a nuclease resistant blocking
moiety, thus protecting extension products from nuclease
degradation. Examples of suitable blocking moieties and
nuclease-mediated approaches are known in the art, and are
described for example in Chen et al., U.S. Pat. No. 7,208,278,
Greenfield et al., U.S. patent application Ser. No. 10/202,211, and
Barany et al., U.S. Pat. No. 6,797,470.
[0026] As used herein, the term "amplifying" refers to any process
that increases the amount of a desired nucleic acid. Any of a
variety of known amplification procedures can be employed in the
present teachings, including PCR (see for example U.S. Pat. No.
4,683,202), as well as any of a variety of ligation-mediated
approaches, including LDR and LCR (see for example U.S. Pat. No.
5,494,810, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,054,564). Some
other amplification procedures include isothermal approaches such
as rolling circle amplification and helicase-dependant
amplification.
[0027] As used herein, the term "blocking complex" refers to a
structure formed by the interaction of a methylated cytosine with a
MeDNA binding protein, as well as the structure formed by the
interaction of an unmethylated cytosine with a non-MeDNA binding
protein. Both situations bring about the inability of a primer
extension reaction to proceed given the presence of the blocking
complex.
[0028] As used herein, the term "cytosine position of interest"
refers to a cytosine residue in a nucleic acid whose methylation
status is relevant to the experimentalist.
[0029] As used herein, the term "near a primer binding site" and
various usages of it, refers to the location of a cytosine of
interest in a nucleic acid, in reference to the position of a first
primer. Thus, a cytosine of interest can be in the sequence of the
primer binding site, or can be 1, 2 or fewer, 3 or fewer, 4 or
fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer,
10 or fewer, 11 or fewer, 12 or fewer, 13 or fewer, 14 or fewer, 15
or fewer, 16 or fewer, 17 or fewer, 18 or fewer, 19 or fewer, 20 or
fewer, 21 or fewer, 22 or fewer, 23 or fewer, 24 or fewer, 25 or
fewer, 26 or fewer, 27 or fewer, 28 or fewer, 29 or fewer, 30 or
fewer, 30-40, 40-50, 50-60, 60-70, 70-100, 100-150, 150-300,
300-500, or 500-1000, nucleotides away from the 3' end of the first
primer. The various embodiments depicted in FIGS. 3 and 6
illustrate situations where the cytosine position of interest is
near a primer binding site.
[0030] As used herein, the term "cofactor" refers to compounds that
binds to an enzyme to facilitate enzyme catalysis. In those
embodiments employing a MeDNA binding protein, cofactors serve the
function of modulate the binding of the protein to DNA. In those
embodiments employing a non-MeDNA binding protein, cofactors serve
the function of providing transferable chemical motif for the
enzyme reaction.
[0031] As used herein, the term "first primer" refers generally to
the primer employed in the extension reaction. A first control
specific primer is one example of a first primer. A first control
specific primer hybridizes to a "first control specific primer
site." A first target specific primer is another example of a first
primer. A first target specific primer hybridizes to a "first
target specific primer site".
[0032] As used herein, the term "second primer" refers generally to
a primer employed in a PCR, which hybridizes to the extension
product produced in the extension reaction, and which can extend to
form a complementary strand. In those embodiments in which the
amplifying is a PCR, a first primer can hybridize to the
complementary strand generated by extension of the second primer,
and itself become extended to effectuate the PCR process. A second
control specific primer is one example of a second primer. A second
control primer can hybridize to a "control specific extension
product". A second target specific primer is another example of a
second primer. A second target specific primer hybridizes to a
"target specific extension product".
[0033] As used herein, the term "extension product" refers
generally to the result of a primer extension reaction. A target
specific extension product is one example of an extension product,
it resulting from extension of a first target specific primer. A
control specific extension product is another example of an
extension product, it resulting from extension of a first control
specific primer.
[0034] As used herein, the term "MeDNA binding protein" refers to a
protein that binds methylated cytosine in a nucleic acid, thus
forming a binding complex. Examples of MeDNA binding proteins
include MeCP2, MBD1, MBD2, MBD3 and MBD4 (Fraga, M F, et al Nucleic
Acid Research, 2003, 31(6), 1765-1774), Dnmt1 (Cheng, X. and
Blumenthal R M Structure, 2008, 16, 341-350), methyl-CpG
antibodies, recombinant proteins with multiple methyl-DNA-binding
domains (Jorgensen, H F. Et al Nucleic Acid Research, 2006, 34(13),
e96), McrBC, and VIM1 (Woo, H R et al Genes & Development,
2007, 21, 267-277).
[0035] As used herein, the term "non MeDNA binding protein" refers
to a protein that binds unmethylated cytosine in a nucleic acid,
thus forming a binding complex. Examples of non-MeDNA biding
proteins include Dnmt3a and Dnmt3b ((Cheng, X. and Blumenthal R M
Structure, 2008, 16, 341-350), M. Sssl (Flynn, J. et al
Biochemistry, 1996, 35, 7308-7315), CXXC domain of MDB1 (Voo, K S
et al Mol. Cell Biol. 2000, 20, 2108-2021).
[0036] As used herein, the term "Ct value" refers to a cycle in a
PCR at which a particular intensity of a probe is observed.
Examples of PCR analysis using Ct values can be found in U.S. Pat.
No. 7,132,239, U.S. Pat. No. 7,057,025, U.S. Pat. No. 6,890,718,
U.S. Pat. No. 5,952,202, U.S. Pat. No. 6,884,583, and U.S. Pat. No.
6,432,642.
MeDNA binding Protein Embodiments
[0037] In some embodiments, the present teachings provide a method
of quantitating methylation in a target nucleic acid. For example,
in some embodiments, the present teachings provide a method of
quantitating methylation in a target nucleic acid comprising;
treating a target nucleic acid with a MeDNA binding protein,
wherein the MeDNA binding protein forms a blocking complex with a
methylated cytosine in the target nucleic acid, wherein the
methylated cytosine in the target nucleic acid is near a first
target specific primer binding site; extending a first target
specific primer hybridized to the first target specific primer
binding site to form a target nucleic acid extension product;
degrading the target nucleic acid; amplifying the target nucleic
acid extension product; determining the difference between the
amount of the target nucleic acid with the amount of a control
nucleic acid lacking a methylated cytosine; and, quantitating
methylation in the target nucleic acid.
[0038] In some embodiments, the amplifying comprises a polymerase
chain reaction comprising a first target specific primer and a
second target specific primer.
[0039] In some embodiments, the degrading comprises treating the
target nucleic acid with a nuclease, wherein the target nucleic
acid extension product is resistant to the nuclease due to a
blocking moiety in the first target specific primer.
[0040] One illustrative embodiment is depicted in FIG. 1. Here, a
target nucleic acid (1) containing a methylated cytosine residue
(M) is present in a reaction mixture. A control nucleic acid (2)
contains an unmethylated cytosine (C). The target nucleic acid can
be treated with a MeDNA binding protein (oval shape), which can
form a blocking complex with a methylated cytosine in the target
nucleic acid (3). The control nucleic acid, lacking a methylated
cytosine, fails to form a blocking complex with the MeDNA binding
protein (note that the oval, representing the MeDNA binding
protein, is not bound with the unmethylated cytosine (C) in the
control nucleic acid (2). A primer extension reaction can be
attempted by hybridizing a first target specific primer (30) to the
target nucleic acid, and a first control specific primer (31) to
the control nucleic acid. The target nucleic acid with the
methylated cytosine, due to the blocking complex with the MeDNA
binding protein, is unable to undergo primer extension (4),
producing for example truncated extension products (6). However,
the control nucleic acid with the unmethylated cytosine is able to
undergo primer extension (5) due to the absence of a blocking
complex, thus forming a control nucleic acid extension product
(dashed, (7)). The target nucleic acid and control nucleic can then
be degraded (note the absence of (1) and (2)), leaving extension
products in tact. Amplifying the extension products can then be
performed, here shown by a PCR using a first target specific primer
(8) and a second target specific primer (9) for the target nucleic
acid, and a first control specific primer (10) and a second control
specific primer (11) for the control nucleic acid. Determining the
difference between the amount of the target nucleic acid with the
amount of a control nucleic acid lacking a methylated cytosine
allows for quantitating methylation in the target nucleic acid.
Here, amplification can proceed on the control extension product,
but fails on the truncated target extension product.
[0041] Illustrative graphs arising from practice of the method of
FIG. 1, are depicted in FIG. 2. FIG. 2A shows the hypothetical
results of an experiment in which the target nucleic acid is more
methylated than the control nucleic acid. Here, the results of a
real-time PCR illustrate that the Ct value for the control nucleic
acid (12) is lower (left-shifted) than the Ct value for the target
nucleic acid (13). This shift reflects the greater number of
extension products resulting from the extension reaction of the
control nucleic acid as compared to the target nucleic acid. This
can be interpreted to reflect that the MeDNA binding protein
preferentially bound target nucleic acids containing methylated
cytosine to form blocking complexes, thus preventing the formation
of target nucleic acid extension products. Thus, the target nucleic
acids contain a greater amount of methylated cytosine at the
cytosine position of interest than do the control nucleic
acids.
[0042] FIG. 2B shows the hypothetical results of an experiment in
which the target nucleic acid is less methylated than the control
nucleic acid. Here, the results of a real-time PCR illustrate that
the Ct value for the target nucleic acid (14) is lower
(left-shifted) than the Ct value for the control nucleic acid (15).
This shift reflects the greater number of extension products
resulting from the extension reaction of the target nucleic acid as
compared to the control nucleic acid. This can be interpreted to
reflect that the MeDNA binding protein preferentially bound control
nucleic acids containing methylated cytosine to form blocking
complexes, thus preventing the formation of control nucleic acid
extension products. Thus, the target nucleic acids contain a lesser
amount of methylated cytosine at the cytosine position of interest
than do the control nucleic acids.
[0043] FIG. 3 illustrates some various possible relationships
between the location of a cytosine of interest, the blocking
complex formed with the MeDNA binding protein, and the first
primer. In some embodiments, the first primer hybridizes upstream
from the blocking complex formed by the MeDNA binding protein and
the methylated cytosine in the target nucleic acid, and further
extension of the first primer is blocked. This is depicted in FIG.
3A. Note that in FIG. 3A the first primer is shown hybridized (see
vertical lines indicating Hydrogen bonds) upstream from the
cytosine of interest, and that the primer is partially extended
(dashed horizontal line). However, the blocking complex between the
methylated cytosine and the MeDNA binding protein blocks further
extension of the first primer. In some embodiments, the first
primer hybridizes on the methylated cytosine on the blocking
complex formed by the MeDNA binding protein and the methylated
cytosine in the target nucleic acid, and initial extension of the
first primer is blocked. This is depicted in FIG. 3B. Note than in
FIG. 3B the first primer is shown hybridized (see the vertical
lines indicating Hydrogen bonds) on the cytosine of interest, and
that the primer is not extended. The blocking complex between the
methylated cytosine and the MeDNA binding protein blocks initial
extension of the first primer. In some embodiments, the first
primer does not hybridize to the methylated cytosine on the
blocking complex formed by the MeDNA binding protein and the
methylated cytosine in the target nucleic acid. This is depicted in
FIG. 3C. Note the absence of vertical lines between the first
primer and the target nucleic acid, indicating that the blocking
complex formed by the MeDNA binding protein and the methylated
cytosine in the target nucleic acid prevents the first primer from
hybridizing. As will be appreciated by one of ordinary skill in the
art in light of the present teachings, the choice of position
between the cytosine of interest, the MeDNA binding protein, and
the primer binding site can be chosen according to the
experimentalist using routine experimentation.
[0044] The control nucleic acid can be employed in a variety of
ways. For example, the control nucleic acid can be in the same
reaction mixture as the target nucleic acid and can be a different
sequence than the target nucleic acid. The control nucleic acid can
be of a known concentration, and can be known to contain an
unmethylated cytosine or a particular amount of unmethylated
cytosine at the position of interest. In some embodiments, the
control nucleic acid can be in a different reaction mixture from
the target nucleic acid. For example, the control nucleic acid can
be the same sequence as the target nucleic acid, and can be present
in a known amount in the different reaction mixture. In some
embodiments, the control nucleic acid can be a different sequence
than the target nucleic acid, and can be present in a known amount
in the different reaction mixture. Various methods of performing
the control reactions will be appreciated by one of skill in the
art in light of the present teachings, including for example
employing controls of the appropriate abundance class (see Bodeau
et al., U.S. patent application Ser. No. 11/372,242.
[0045] The present teachings also provide a method of quantitating
methylation in a target nucleic acid comprising;
[0046] treating, in any order, [0047] (a) the target nucleic acid
with a MeDNA binding protein, wherein the MeDNA binding protein
forms a blocking complex with a methylated cytosine in the target
nucleic acid, wherein the methylated cytosine is disposed between a
first target specific primer binding site and a second target
specific primer binding site; and, [0048] (b) a control nucleic
acid with a MeDNA binding protein, wherein the MeDNA binding
protein fails to form a blocking complex with an unmethylated
cytosine in the control nucleic acid, wherein the non-methylated
cytosine is disposed between a first control specific primer
binding site and a second control specific primer binding site;
[0049] extending, in any order, [0050] (a) a first target specific
primer hybridized to the first target specific primer binding site
to form a target nucleic acid extension product; and, [0051] (b) a
first control specific primer hybridized to the first control
specific primer binding site to form a control nucleic acid
extension product; [0052] degrading, in any order, [0053] (a) the
target nucleic acid; and, [0054] (b) the control nucleic acid;
[0055] amplifying, in any order, [0056] (a) the target nucleic acid
extension product in a polymerase chain reaction comprising a first
target specific primer and a second target specific primer; and,
[0057] (b) the control nucleic acid extension product in a
polymerase chain reaction comprising a first control specific
primer and a second control specific primer;
[0058] determining the difference between the amount of target
nucleic acid with the amount of control nucleic acid; and,
[0059] quantitating methylation in the target nucleic acid.
[0060] In some embodiments, the determining comprises;
[0061] measuring, in any order, [0062] (a) a first Ct value
associated with the amount of the target nucleic acid, and, [0063]
(b) a second Ct value associated with the amount of the control
nucleic acid; and,
[0064] quantitating methylation in the target nucleic by comparing
the first Ct value with the second Ct value. [0065] In some
embodiments, the first Ct value is higher than the second Ct value,
and the target nucleic acid is more methylated than the control
nucleic acid.
[0066] In some embodiments, the first Ct value is lower than the
second Ct value, and the target nucleic acid is less methylated
than the control nucleic acid.
[0067] In some embodiments, the target nucleic acid and the control
nucleic acid comprise the same first primer binding site and the
same second primer binding site.
[0068] In some embodiments, the target nucleic acid is amplified in
a separate reaction vessel from the control nucleic acid.
[0069] In some embodiments, the target nucleic acid and the control
nucleic acid comprise the same first primer binding site and the
same second primer binding site and are amplified with a common
first primer and a common second primer.
[0070] In some embodiments, the target nucleic acid is amplified in
a same reaction vessel as the control nucleic acid.
[0071] In some embodiments, the target nucleic acid and the control
nucleic acid comprise a different first primer binding site and a
different second primer binding site and are amplified with a
different first primer and a different second primer.
[0072] In some embodiments, the quantitating comprises measuring an
interchelating dye.
[0073] In some embodiments, the determining comprises;
[0074] measuring displacement of a target sequence specific probe,
wherein the target sequence specific probe hybridizes to a region
of the target nucleic acid extension product, or complement to the
target nucleic acid extension product, disposed between the first
target specific primer binding site and the second target specific
primer binding site;
[0075] measuring displacement of a control sequence specific probe,
wherein the control sequence specific probe hybridizes to a region
of the control nucleic acid extension product, or complement to the
control nucleic acid extension product, disposed between the first
control specific primer binding site and the second control
specific primer binding site.
[0076] In some embodiments, the treating with the MeDNA binding
protein further comprises a cofactor.
[0077] In some embodiments, the cofactor is SELECTED FROM THE GROUP
CONSISTING OF S-adenosylmethionine, S-adenosylhomocysteine and
sinefungin. In a preferred embodiment, S-adenosylhomocycteine is
used.
[0078] In some embodiments, the first primer hybridizes upstream
from the blocking complex formed by the MeDNA binding protein and
the methylated cytosine in the target nucleic acid, and further
extension of the first primer is blocked.
[0079] In some embodiments, the first primer hybridizes on the
methylated cytosine on the blocking complex formed by the MeDNA
binding protein and the methylated cytosine in the target nucleic
acid, and initial extension of the first primer is blocked.
[0080] In some embodiments, the first primer does not hybridize to
the methylated cytosine on the blocking complex formed by the MeDNA
binding protein and the methylated cytosine in the target nucleic
acid.
Non-MeDNA Binding Protein Embodiments
[0081] In some embodiments, the present teachings provide a method
of quantitating methylation in a target nucleic acid. For example,
in some embodiments, the present teachings provide a method of
quantitating methylation in a target nucleic acid comprising;
treating a control nucleic acid with a non-MeDNA binding protein,
wherein the non-MeDNA binding protein forms a blocking complex with
an unmethylated cytosine in the control nucleic acid, wherein the
unmethylated cytosine in the control nucleic acid is near a first
control specific primer binding site; extending a first primer
hybridized to the first primer binding site to form a control
nucleic acid extension product; degrading the control nucleic acid;
amplifying the control nucleic acid extension product; determining
the difference between the amount of the control nucleic acid with
the amount of a target nucleic acid containing a methylated
cytosine; and, quantitating methylation in the target nucleic
acid.
[0082] In some embodiments, the amplifying comprises a polymerase
chain reaction comprising a first target specific primer and a
second target specific primer.
[0083] In some embodiments, the degrading comprises treating the
target nucleic acid with a nuclease, wherein the target nucleic
acid extension product is resistant to the nuclease due to a
blocking moiety in the first target specific primer.
[0084] One illustrative embodiment is depicted in FIG. 4. Here, a
target nucleic acid (16) containing a methylated cytosine residue
(M) is present in a reaction mixture. A control nucleic acid (17)
contains an unmethylated cytosine (C). The target nucleic acid can
be treated with a non-MeDNA binding protein (oval shape), but due
to the presence of a methyl group on the cytosine of interest,
fails to form a blocking complex with the non-MeDNA binding protein
(note that the oval shape, representing the non-MeDNA binding
protein, is not bound with the methylated cytosine (C) in the
target nucleic acid (16)). The control nucleic acid, lacking a
methylated cytosine, is able to form a blocking complex (18) with
the non-MeDNA binding protein (note that the oval, representing the
MeDNA binding protein, is bound with the unmethylated cytosine (C)
in the control nucleic acid (17). A primer extension reaction can
be performed. The control nucleic acid with the unmethylated
cytosine, due to the blocking complex with the non-MeDNA binding
protein, is unable to undergo primer extension (19) with a first
primer (33), producing for example truncated extension products
(20). However, the target nucleic acid with the methylated cytosine
is able to undergo primer extension with a first primer (32), thus
forming a target nucleic acid extension product (dashed, (21)). The
target nucleic acid and control nucleic can then be degraded (note
the absence of (16) and (17), leaving behind any extension
products. Amplifying the extension products can then be performed,
here shown as a PCR using a first target specific primer (22) and a
second target specific primer (23) for the target nucleic acid, and
a first control specific primer (24) and a second control specific
primer (25) for the control nucleic acid. Determining the
difference between the amount of the target nucleic acid with the
amount of a control nucleic acid lacking a methylated cytosine
allows for quantitating methylation in the target nucleic acid.
Here, amplification can proceed on the target extension product,
but fails on the truncated control extension product.
[0085] Representative graphs arising from practicing the method of
FIG. 4, are depicted in FIG. 5. FIG. 5A shows the results of an
experiment in which the target nucleic acid is more methylated than
the control nucleic acid. Here, the results of a real-time PCR
illustrate that the Ct value for the control nucleic acid (26) is
higher (right-shifted) than the Ct value for the target nucleic
acid (27). This shift reflects the greater number of extension
products resulting from the extension reaction of the methylated
target nucleic acid as compared to the control nucleic acid. This
can be interpreted to reflect that the non-MeDNA binding protein
preferentially bound control nucleic acids containing unmethylated
cytosine, thus preventing the formation of control nucleic acid
extension products. Thus, the target nucleic acids contain a
greater amount of methylated cytosine at the cytosine position of
interest than do the control nucleic acids.
[0086] FIG. 5B shows the results of an experiment in which the
target nucleic acid is less methylated than the control nucleic
acid. Here, the results of a real-time PCR illustrate that the Ct
value for the control nucleic acid (28) is lower (left-shifted)
than the Ct value for the target nucleic acid (29). This shift
reflects the greater number of extension products resulting from
the extension reaction of the control nucleic acid as compared to
the target nucleic acid. This can be interpreted to reflect that
the non-MeDNA binding protein preferentially bound target nucleic
acids containing unmethylated cytosine, thus preventing the
formation of target nucleic acid extension products. Thus, the
target nucleic acids contain a lesser amount of methylated cytosine
at the cytosine position of interest than do the control nucleic
acids.
[0087] FIG. 6 illustrates the various possible relationships
between the location of a cytosine of interest, the blocking
complex formed with the MeDNA binding protein, and the first
primer. In some embodiments, the first primer hybridizes upstream
from the blocking complex formed by the non-MeDNA binding protein
and the unmethylated cytosine in the target nucleic acid, and
further extension of the first primer is blocked. This is depicted
in FIG. 6A. Note that in FIG. 6A the first primer is shown
hybridized (see vertical lines indicating Hydrogen bonds) upstream
from the cytosine of interest, and that the primer is partially
extended (dashed horizontal line). However, the blocking complex
between the unmethylated cytosine and the non-MeDNA binding protein
blocks further extension of the first primer. In some embodiments,
the first primer hybridizes on the unmethylated cytosine on the
blocking complex formed by the non-MeDNA binding protein and the
unmethylated cytosine in the target nucleic acid, and initial
extension of the first primer is blocked. This is depicted in FIG.
6B. Note than in FIG. 6B the first primer is shown hybridized (see
the vertical lines indicating Hydrogen bonds) on the cytosine of
interest, and that the primer is not extended. The blocking complex
between the unmethylated cytosine and the non-MeDNA binding protein
blocks initial extension of the first primer. In some embodiments,
the first primer does not hybridize to the unmethylated cytosine on
the blocking complex formed by the non-MeDNA binding protein and
the unmethylated cytosine in the target nucleic acid. This is
depicted in FIG. 6C. Note the absence of vertical lines between the
first primer and the target nucleic acid, indicating that the
blocking complex formed by the non-MeDNA binding protein and the
unmethylated cytosine in the target nucleic acid prevents the first
primer from hybridizing. As will be appreciated by one of ordinary
skill in the art in light of the present teachings, the choice of
position between the cytosine of interest, the non-MeDNA binding
protein, and the primer binding site can be chosen according to the
experimentalist using routine experimentation.
[0088] In view of FIG. 4, the control nucleic acid can be employed
in a variety of ways. For example, the control nucleic acid can be
in the same reaction mixture as the target nucleic acid and can be
a different sequence than the target nucleic acid. The control
nucleic acid can be of a known concentration, and can be known to
contain an unmethylated cytosine at the position of interest. In
some embodiments, the control nucleic acid can be in a different
reaction mixture from the target nucleic acid. For example, the
control nucleic acid can be the same sequence as the target nucleic
acid, and can be present in a known amount in the different
reaction mixture. In some embodiments, the control nucleic acid can
be a different sequence than the target nucleic acid, and can be
present in a known amount in the different reaction mixture.
Various methods of performing the control reactions will be
appreciated by one of skill in the art in light of the present
teachings, including for example employing controls of the
appropriate abundance class (see Bodeau et al., U.S. patent
application Ser. No. 11/372,242).
[0089] The present teachings also provide a method of quantitating
methylation in a target nucleic acid comprising;
[0090] treating, in any order, [0091] (a) the control nucleic acid
with a non-MeDNA binding protein, wherein the non-MeDNA binding
protein forms a blocking complex with an unmethylated cytosine in
the control nucleic acid lacking, wherein the unmethylated cytosine
is disposed between a first control-specific primer binding site
and a second control-specific primer binding site; and, [0092] (b)
a target nucleic acid with a non-MeDNA binding protein, wherein the
non-MeDNA binding protein fails to form a blocking complex with a a
methylated cytosine in the target nucleic acid, wherein the
methylated cytosine is disposed between a first target specific
primer binding site and a second target specific primer binding
site;
[0093] extending, in any order, [0094] (a) a first target specific
primer hybridized to the first target specific primer binding site
to form a target nucleic acid extension product; and, [0095] (b) a
first control specific primer hybridized to the first control
specific primer binding site to form a control nucleic acid
extension product;
[0096] degrading, in any order, [0097] (a) the target nucleic acid;
and, [0098] (b) the control nucleic acid;
[0099] amplifying, in any order, [0100] (a) the target nucleic acid
extension product in a polymerase chain reaction comprising a first
target specific primer and a second target specific primer; and,
[0101] (b) the control nucleic acid extension product in a
polymerase chain reaction comprising a first control specific
primer and a second control specific primer;
[0102] determining the difference between the amount of target
nucleic acid with the amount of control nucleic acid; and,
[0103] quantitating methylation in the target nucleic acid.
[0104] In some embodiments, the determining comprises;
[0105] measuring, in any order, [0106] (a) a first Ct value
associated with the amount of the target nucleic acid, and, [0107]
(b) a second Ct value associated with the amount of the control
nucleic acid; and,
[0108] quantitating methylation in the target nucleic by comparing
the first Ct value with the second Ct value.
[0109] In some embodiments, the first Ct value is higher than the
second Ct value, and the target nucleic acid is less methylated
than the control nucleic acid.
[0110] In some embodiments, the first Ct value is lower than the
second Ct value, and the target nucleic acid is more methylated
than the control nucleic acid.
[0111] In some embodiments, the target nucleic acid and the control
nucleic acid comprise the same first primer binding site and the
same second primer binding site.
[0112] In some embodiments, the target nucleic acid is amplified in
a separate reaction vessel from the control nucleic acid.
[0113] In some embodiments, the target nucleic acid and the control
nucleic acid comprise the same first primer binding site and the
same second primer binding site and are amplified with a common
first primer and a common second primer.
[0114] In some embodiments, the target nucleic acid is amplified in
a same reaction vessel as the control nucleic acid.
[0115] In some embodiments, the target nucleic acid and the control
nucleic acid comprise a different first primer binding site and a
different second primer binding site and are amplified with a
different first primer and a different second primer.
[0116] In some embodiments, the quantitating comprises measuring an
interchelating dye.
[0117] In some embodiments, the determining comprises;
[0118] measuring displacement of a target sequence specific probe,
wherein the target sequence specific probe hybridizes to a region
of the target nucleic acid extension product, or complement to the
target nucleic acid extension product, disposed between the first
target specific primer binding site and the second target specific
primer binding site;
[0119] measuring displacement of a control sequence specific probe,
wherein the control sequence specific probe hybridizes to a region
of the control nucleic acid extension product, or complement to the
control nucleic acid extension product, disposed between the first
control specific primer binding site and the second control
specific primer binding site.
[0120] In some embodiments, the treating with the non-MeDNA binding
protein further comprises a cofactor.
[0121] In some embodiments, the cofactor is selected from the group
consisting of S-adenosylmethionine, S-adenosylhomocysteine and
sinefungin. In a preferred embodiment, S-adenosylhomocycteine is
used.
[0122] In some embodiments, the first primer hybridizes upstream
from the blocking complex formed by the non-MeDNA binding protein
and the unmethylated cytosine in the control nucleic acid, and
further extension of the first primer is blocked.
[0123] In some embodiments, the first primer hybridizes on the
unmethylated cytosine on the blocking complex formed by the
non-MeDNA binding protein and the unmethylated cytosine in the
control nucleic acid, and initial extension of the first primer is
blocked.
[0124] In some embodiments, the first primer does not hybridize to
the unmethylated cytosine on the blocking complex formed by the
non-MeDNA binding protein and the unmethylated cytosine in the
target nucleic acid.
Kits
[0125] The instant teachings also provide kits designed to expedite
performing certain of the disclosed methods. Kits may serve to
expedite the performance of certain disclosed methods by assembling
two or more components required for carrying out the methods. In
certain embodiments, kits contain components in pre-measured unit
amounts to minimize the need for measurements by end-users. In some
embodiments, kits include instructions for performing one or more
of the disclosed methods. Preferably, the kit components are
optimized to operate in conjunction with one another.
MeDNA Binding Protein Kits
[0126] In some embodiments, the present teachings provide a kit for
quantitating methylation in a target nucleic acid comprising;
[0127] a Me-DNA binding protein;
[0128] a first target specific primer;
[0129] a second target specific primer;
[0130] a first control specific primer;
[0131] a second control specific primer; and,
[0132] a polymerase.
[0133] In some embodiments of the kit,
[0134] the first control specific primer is a different sequence
from the first target specific primer; and,
[0135] the second control specific primer is a different sequence
from the second target specific primer.
[0136] In some embodiments of the kit,
[0137] the first control specific primer is a same sequence as the
first target specific primer; and,
[0138] the second control specific primer is a same sequence as the
second target specific primer.
[0139] In some embodiments, the kit further comprises a control
sequence specific probe.
[0140] In some embodiments, the kit further comprises a target
sequence specific probe, wherein the control sequence specific
probe is a different sequence from the target sequence specific
probe.
[0141] In some embodiments, the kit further comprises a target
sequence specific probe, wherein the control sequence specific
probe is a same sequence as the target sequence specific probe.
[0142] In some embodiments, the kit comprises a cofactor.
[0143] Non-MeDNA Binding Protein Kits
[0144] In some embodiments, the present teachings provide a kit for
quantitating methylation in a target nucleic acid comprising;
[0145] a non-MeDNA binding protein;
[0146] a first target specific primer;
[0147] a second target specific primer;
[0148] a first control specific primer;
[0149] a second control specific primer; and,
[0150] a polymerase.
[0151] In some embodiments of the kit,
[0152] the first control specific primer is a different sequence
from the first target specific primer; and,
[0153] the second control specific primer is a different sequence
from the second target specific primer.
[0154] In some embodiments, the first control specific primer is a
same sequence as the first target specific primer; and, [0155] the
second control specific primer is a same sequence as the second
target specific primer.
[0156] In some embodiments, the kit comprises a control sequence
specific probe.
[0157] In some embodiments, the kit comprises a target sequence
specific probe, wherein the control sequence specific probe is a
different sequence from the target sequence specific probe.
[0158] In some embodiments, the kit comprises a target sequence
specific probe, wherein the control sequence specific probe is a
same sequence as the target sequence specific probe.
[0159] In some embodiments, the kit comprises a cofactor.
[0160] Although the disclosed teachings have been described with
reference to various applications, methods, and kits, it will be
appreciated that various changes and modifications may be made
without departing from the teachings herein. The foregoing examples
are provided to better illustrate the present teachings and are not
intended to limit the scope of the teachings herein. Certain
aspects of the present teachings may be further understood in light
of the following claims.
* * * * *