U.S. patent application number 17/220658 was filed with the patent office on 2021-07-22 for determination of nucleic acid methylation.
The applicant listed for this patent is Agency for Science, Technology and Research. Invention is credited to Jackie Y. Ying, Yanbing Zu.
Application Number | 20210222237 17/220658 |
Document ID | / |
Family ID | 1000005495049 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210222237 |
Kind Code |
A1 |
Zu; Yanbing ; et
al. |
July 22, 2021 |
DETERMINATION OF NUCLEIC ACID METHYLATION
Abstract
The present invention relates to methods and kits for
determining the methylation status of a target nucleic acid
molecule in a sample.
Inventors: |
Zu; Yanbing; (Singapore,
SG) ; Ying; Jackie Y.; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agency for Science, Technology and Research |
Singapore |
|
SG |
|
|
Family ID: |
1000005495049 |
Appl. No.: |
17/220658 |
Filed: |
April 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15778876 |
May 24, 2018 |
11021743 |
|
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PCT/SG2016/050583 |
Nov 25, 2016 |
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17220658 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2563/155 20130101;
C12Q 2537/163 20130101; C12Q 2535/131 20130101; C12Q 1/6858
20130101; C12Q 2600/154 20130101; C12Q 1/6886 20130101; C12Q
2565/107 20130101; C12Q 2523/125 20130101; C12Q 2525/113
20130101 |
International
Class: |
C12Q 1/6858 20060101
C12Q001/6858; C12Q 1/6886 20060101 C12Q001/6886 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 27, 2015 |
SG |
10201509792S |
Claims
1. A method of determining the methylation status of a target
nucleic acid molecule in a sample, wherein the method comprises the
steps of: (a) bisulfate treatment of the sample for converting the
unmethylated cytosine bases in the target nucleic acid molecule to
uracil; (b) amplifying at least part of the converted target
nucleic acid molecule, said part comprising a target region the
methylation status of which is to be analyzed, in a polymerase
chain reaction (PCR), using a pair of methylation-specific primers
under conditions allowing such amplification to generate PCR
amplification products (amplicons); (c) contacting the amplicons
with a methylation-specific plasmonic nanoprobe comprising a
plasmonic nanoparticle and a non-ionic oligonucleotide analog probe
covalently coupled thereto, the oligonucleotide analog probe
comprising a base sequence that is complementary to the target
region, under conditions that allow the oligonucleotide analog
probe and the amplicons comprising the target region to hybridize
to each other, wherein the probe generates a detectable signal if
hybridized to the target nucleic acid molecule that is
distinguishable from the signal of the unhybridized probe; (d)
determining the methylation status of the target nucleic acid
molecule based on the determination of the melting temperature
T.sub.m of the hybrid of the nanoprobe and the target nucleic
acid.
2. The method according to claim 1, wherein the methylation status
of one or more CpG islands within the target nucleic acid molecule
is determined.
3. The method according to claim 1, wherein asymmetric PCR (aPCR)
is used in step (b) of the method.
4. The method according to claim 1, wherein at least one of the
methylation-specific primers hybridizes to the converted target
nucleic acid molecule at a region comprising one or more CpG
islands, such that the primer pair preferably hybridizes to the
converted methylated nucleic acid molecule over the converted
unmethylated molecule during the PCR.
5. The method according to claim 1, wherein a blocker sequence is
added during the PCR to minimize the amplification of the converted
unmethylated nucleic acid molecule.
6. The method according to claim 1, wherein the plasmonic
nanoparticle used in step (c) of the method is a plasmonic gold
nanoparticle.
7. The method according to claim 1, wherein the non-ionic
oligonucleotide analog probe used in step (c) of the method is a
morpholino oligonucleotide probe.
8. The method according to claim 1, wherein the detectable signal
in step (c) of the method is the color of the assay solution that
is indicative of whether the probe is hybridized to the amplicons
or not.
9. The method according to claim 1, wherein the melting temperature
in step (d) of the method is indicated by a color change caused by
nanoprobe dissociation and subsequent aggregation.
10. The method according to claim 1, wherein the method further
comprises isolating genomic DNA from the sample prior to step (a)
of the method.
11. The method according to claim 1, wherein the method further
comprises using a nucleic acid molecule comprising the unmethylated
target nucleotide sequence as a negative control, and/or using a
nucleic acid molecule comprising the methylated target nucleotide
sequence as a positive control.
12. The method according to claim 1, wherein the target nucleic
acid molecule is the human Septin 9 (SEPT9) gene promoter, the
methylation-specific primers have the nucleic acid sequences
5'-ATTAGTTATTATGTCGGATTTCGC-3' (SEQ ID NO: 1) and
5'-CAACACGTCCGCGACCG-3' (SEQ ID NO: 2), the blocker has the nucleic
acid sequence 5'-GTTATTATGTTGGATTTTGTGGTTAATGTGTAG-3' and is
labelled with C3 spacer at the 3' end (SEQ ID NO: 3), and the
oligonucleotide analog probe used is a morpholino oligonucleotide
having the nucleic acid sequence 5'-CAACTACGCGTTAACCGCGATTTTT-3'
(SEQ ID NO: 4).
13. The method according to claim 12, wherein the method is used to
determine the methylation status of the human Septin 9 (SEPT9) gene
promoter, preferably in the cell-free DNA (cfDNA) fragments
circulating in the blood released from colorectal cancer cells.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to methods and kits
for detection of DNA methylation.
BACKGROUND OF THE INVENTION
[0002] Epigenetic modifications of human genes are emerging as
promising biomarkers for early diagnosis of cancer. In particular,
DNA methylation of the promoter regions of cancer suppression genes
is usually associated with aberrant gene regulation involved in the
predisposition, initiation, and progression of cancer. Therefore,
the DNA methylation biomarkers could be ideal targets for the early
detection of cancer and for the monitoring of cancer
progression.
[0003] For example, recent studies showed that blood-based
detection of Septin 9 (SEPT9) gene methylation may be used as a
non-invasive method for colorectal cancer screening. The
circulating cell-free DNA (cfDNA) fragments released from tumor
cells allow for early detection of the aberrant DNA alteration. It
has been reported that the clinical sensitivity and specificity of
the SEPT9 test could be higher than 70%. Compared to the
colonoscopic-based surveillance, the DNA assay is much more
convenient and should improve patient compliance to the screening.
However, to gauge the epigenetic change of the rare circulating
tumor cfDNA in a large background of normal DNA is very
challenging. Highly sensitive and specific detection is
required.
[0004] Various technologies have been existing in the prior art for
the determination of nucleic acid methylation. However, there still
remains a considerable need for new technologies to overcome the
drawbacks of existing technologies.
SUMMARY OF THE INVENTION
[0005] The present invention satisfies the aforementioned need in
the art by providing a new method for determining nucleic acid
methylation.
[0006] In one aspect, the present invention provides a method of
determining the methylation status of a target nucleic acid
molecule in a sample, wherein the method comprises the steps
of:
(a) bisulfite treatment of the sample for converting the
unmethylated cytosine bases in the target nucleic acid molecule to
uracil; (b) amplifying at least part of the converted target
nucleic acid molecule, said part comprising a target region the
methylation status of which is to be analyzed, in a polymerase
chain reaction (PCR), using a pair of methylation-specific primers
under conditions allowing such amplification to generate PCR
amplification products (amplicons); (c) contacting the amplicons
with a methylation-specific plasmonic nanoprobe comprising a
plasmonic nanoparticle and a non-ionic oligonucleotide analog probe
covalently coupled thereto, the oligonucleotide analog probe
comprising a base sequence that is complementary to the target
region, under conditions that allow the oligonucleotide analog
probe and the amplicons comprising the target region to hybridize
to each other, wherein the probe generates a detectable signal if
hybridized to the target nucleic acid molecule that is
distinguishable from the signal of the unhybridized probe; (d)
determining the methylation status of the target nucleic acid
molecule based on the determination of the melting temperature
T.sub.m of the hybrid of the nanoprobe and the target nucleic
acid.
[0007] In various embodiments, the methylation status of one or
more CpG islands within the target nucleic acid molecule is
determined.
[0008] In various embodiments, asymmetric PCR (aPCR) is used in
step (b) of the method disclosed herein.
[0009] In various embodiments, at least one of the
methylation-specific primers hybridizes to the converted target
nucleic acid molecule at a region comprising one or more CpG
islands, such that the primer pair preferably hybridizes to the
converted methylated nucleic acid molecule over the converted
unmethylated molecule during the PCR.
[0010] In various embodiments, a blocker sequence is added during
the PCR to minimize the amplification of the converted unmethylated
nucleic acid molecule.
[0011] In various embodiments, the plasmonic nanoparticle used in
step (c) of the method disclosed herein is a plasmonic gold
nanoparticle.
[0012] In various embodiments, the non-ionic oligonucleotide analog
probe used in step (c) of the method disclosed herein is a
morpholino oligonucleotide probe.
[0013] In various embodiments, the detectable signal in step (c) of
the method disclosed herein is the color of the assay solution that
is indicative of whether the probe is hybridized to the amplicons
or not.
[0014] In various embodiments, the melting temperature in step (d)
of the method disclosed herein is indicated by a color change
caused by nanoprobe dissociation and subsequent aggregation.
[0015] In various embodiments, the method disclosed herein further
comprises isolating genomic DNA from the sample prior to step
(a).
[0016] In various embodiments, the method further comprises using a
nucleic acid molecule comprising the unmethylated target nucleotide
sequence as a negative control, and/or using a nucleic acid
molecule comprising the methylated target nucleotide sequence as a
positive control.
[0017] In various embodiments, the target nucleic acid molecule is
the human Septin 9 (SEPT9) gene promoter, the methylation-specific
primers have the nucleic acid sequences
5'-ATTAGTTATTATGTCGGATTTCGC-3' (SEQ ID NO: 1) and
5'-CAACACGTCCGCGACCG-3' (SEQ ID NO: 2), the blocker has the nucleic
acid sequence 5'-GTTATTATGTTGGATTTTGTGGTTAATGTGTAG-3' and is
labelled with C3 spacer at the 3' end (SEQ ID NO: 3), and the
oligonucleotide analog probe used is a morpholino oligonucleotide
having the nucleic acid sequence 5'-CAACTACGCGTTAACCGCGATTTTT-3'
(SEQ ID NO: 4).
[0018] In various embodiments, the method is used to determine the
methylation status of the human Septin 9 (SEPT9) gene promoter,
preferably in the cell-free DNA (cfDNA) fragments circulating in
the blood released from colorectal cancer cells.
[0019] In another aspect, the invention provides a kit for
determining the methylation status of a target nucleic acid
molecule in a sample, preferably of one or more CpG islands within
said target nucleic acid molecule, wherein the kit comprises a pair
of methylation-specific primers and a methylation-specific
plasmonic nanoprobe for use in a method disclosed herein.
[0020] In various embodiments, the kit further comprises a blocker
sequence for use in the method disclosed herein.
[0021] In various embodiments, the kit further comprises bisulfate
or a salt thereof.
[0022] In various embodiments, the target nucleic acid molecule is
the human SEPT9 gene promoter, and the kit comprises a pair of
methylation-specific primers having the nucleic acid sequences
5'-ATTAGTTATTATGTCGGATTTCGC-3' (SEQ ID NO: 1) and
5'-CAACACGTCCGCGACCG-3' (SEQ ID NO: 2), and a plasmonic gold
nanoparticle functionalized with a morpholino oligonucleotide
having the nucleic acid sequence 5'-CAACTACGCGTTAACCGCGATTTTT-3'
(SEQ ID NO: 4).
[0023] In various embodiments, the kit further comprises a blocker
having the nucleic acid sequence
5'-GTTATTATGTTGGATTTTGTGGTTAATGTGTAG-3' and is labelled with C3
spacer at the 3' end (SEQ ID NO: 3).
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The invention will be better understood with reference to
the detailed description when considered in conjunction with the
non-limiting examples and the accompanying drawings.
[0025] FIG. 1 shows the workflow of the methylation assay described
herein.
[0026] FIG. 2 shows the SEPT9 gene target region and the design of
primers, blocker and probe.
[0027] FIG. 3 shows the melting temperature as a function of target
concentration for methylated and unmethylated systhetic DNA
samples. Error of T.sub.m measurement: .+-.1.degree. C.
[0028] FIG. 4 shows the assay responses towards unmethylated and
methylated gDNA samples.
[0029] FIG. 5 shows the detection of 0.01% of methylated gDNA in
the unmethylated gDNA background.
[0030] FIG. 6 shows the results of the assays for colorectal cell
line samples. The template in the negative control was unmethylated
gDNA (.about.100 ng).
DETAILED DESCRIPTION OF THE INVENTION
[0031] The following detailed description refers to, by way of
illustration, specific details and embodiments in which the
invention may be practiced. These embodiments are described in
sufficient detail to enable those skilled in the art to practice
the invention. Other embodiments may be utilized and structural,
and logical changes may be made without departing from the scope of
the invention. The various embodiments are not necessarily mutually
exclusive, as some embodiments can be combined with one or more
other embodiments to form new embodiments.
[0032] The object of the present invention is to provide a method
of determining the methylation status of a target nucleic acid
molecule in a sample.
[0033] To this end, the inventors of the present invention have
provided such a method employing methylation-specific polymerase
chain reaction (PCR) and plasmonic nanoprobe-based detection.
[0034] In one aspect, disclosed herein is a method of determining
the methylation status of a target nucleic acid molecule in a
sample, preferably of one or more CpG islands within said target
nucleic acid molecule, wherein the method comprises the steps
of:
(a) bisulfite treatment of the sample for converting the
unmethylated cytosine bases in the target nucleic acid molecule to
uracil; (b) amplifying at least part of the converted target
nucleic acid molecule, said part comprising a target region the
methylation status of which is to be analyzed, in a polymerase
chain reaction (PCR), preferably asymmetric PCR (aPCR), using a
pair of methylation-specific primers under conditions allowing such
amplification to generate PCR amplification products (amplicons);
(c) contacting the amplicons with a methylation-specific plasmonic
nanoprobe comprising a plasmonic nanoparticle, preferably a
plasmonic gold nanoparticle, and a non-ionic oligonucleotide
analog, preferably morpholino oligonucleotide (MOR), probe
covalently coupled thereto, the oligonucleotide analog probe
comprising a base sequence that is complementary to the target
region, under conditions that allow the oligonucleotide analog
probe and the amplicons comprising the target region to hybridize
to each other, wherein the probe generates a detectable signal if
hybridized to the target nucleic acid molecule that is
distinguishable from the signal of the unhybridized probe, wherein
said detectable signal is preferably the color of the assay
solution that is indicative of whether the probe is hybridized to
the amplicons or not; (d) determining the methylation status of the
target nucleic acid molecule based on the determination of the
melting temperature T.sub.m of the hybrid of the nanoprobe and the
target nucleic acid, wherein the melting temperature is preferably
indicated by a color change caused by nanoprobe dissociation and
subsequent aggregation.
[0035] In certain embodiments, the method disclosed herein may
further comprise isolating genomic DNA from the sample prior to
step (a).
[0036] The presently described method is used to determine the
methylation status (i.e., the location and/or degree of
methylation) of one or more cytosines of the target nucleic acid
molecule in a sample. In some embodiments, the differences in the
base composition of the target nucleic acid molecule relative to
the canonical unmethylated sequence thereof is used to determine
the extent of methylation.
[0037] In preferred embodiments, the methylation status of one or
more CpG islands within the target nucleic acid molecule is
determined. CpG islands are regions of the genome containing
clusters of CpG dinucleotides. These clusters frequently appear in
the 5' ends of genes. Methylation of CpG islands is known to play a
role in transcriptional silencing in higher organisms. The cytosine
bases of most CpG dinucleotides in the human genome are methylated,
but the cytosine bases in CpG islands are usually unmethylated.
[0038] The terms "nucleic acid molecule", "nucleic acid sequence"
or "oligonucleotide", as used herein, relate to any nucleic acid
molecule in any possible configuration, including single-stranded,
double-stranded configurations or a combination thereof.
[0039] The term "sample" as used herein refers to anything capable
of being analyzed by the methods described herein. In some
embodiments, the sample comprises or is suspected to comprise one
or more nucleic acids capable of analysis by the methods. In
certain embodiments, for example, the samples comprise nucleic
acids (e.g., DNA, RNA, cDNAs, etc.). Samples can include, for
example, cells, blood, semen, saliva, urine, feces, rectal swabs,
and the like. In some embodiments, the samples are "mixture"
samples, which comprise nucleic acids from more than one subject or
individual. In some embodiments, the methods provided herein
comprise purifying the sample or purifying the nucleic acid(s) from
the sample. In some embodiments, the sample is purified nucleic
acid.
[0040] The term "bisulfite treatment" as used herein refers to the
treatment of a sample with bisulfite or a salt thereof (e.g. sodium
bisulfate) for subsequent methylation analysis. Treatment of
nucleic acid molecules (e.g. DNA) with bisulfite converts
unmethylated cytosine residues to uracil, but leaves
5-methylcytosine residues unmodified. Thus, bisulfite treatment
introduces specific changes in the nucleic acid base composition
that depend on the methylation status of individual cytosine
residues. In some embodiments, bisulfate treatment yields
single-nucleotide resolution information about the methylation
status of a segment of DNA.
[0041] Following bisulfate treatment, at least part of the
converted target nucleic acid molecule is amplified by PCR. Any PCR
that may produce single-stranded amplicons for hybridization to the
oligonucleotide analog probe of the methylation-specific plasmonic
nanoprobe may be used in the present method. Such types of PCR
technology include, but are not limited to allele-specific PCR,
assembly PCR, asymmetric PCR, dial-out PCR, digital PCR,
helicase-dependent amplification, hot start PCR,
intersequence-specific PCR (ISSR), inverse PCR, ligation-mediated
PCR, methylation-specific PCR (MSP), miniprimer PCR, multiplex
ligation-dependent probe amplification (MLPA), multiplex-PCR,
nanoparticle-assisted PCR (nanoPCR), nested PCR, overlap-extension
PCR or splicing by overlap extension (SOEing), PAN-AC, reverse
transcription PCR (RT-PCR), solid phase PCR, thermal asymmetric
interlaced PCR (TAIL-PCR), touchdown PCR (step-down PCR), universal
fast walking or transcription-mediated amplification (TMA). Such
techniques are well-known in the art (McPherson, M J and Moller, S
G (2000) PCR (Basics), Springer-Verlag Telos; first edition).
[0042] In preferred embodiments, asymmetric PCR (aPCR) is used.
aPCR is a PCR wherein the amounts of the two primers are unequal.
The primer present at a higher amount is referred to as the excess
primer, and the strand resulting from the extension of the excess
primer is accumulated in excess and is hybridized subsequently to
the oligonucleotide analog probe of the methylation-specific
plasmonic nanoprobe.
[0043] The methylation-specific primers are designed to
specifically amplify the converted methylated target nucleic acid
molecule. In certain embodiments, at least one of the
methylation-specific primers hybridizes to the converted target
nucleic acid molecule at a region comprising one or more CpG
islands, such that the primer pair preferably hybridizes to the
converted methylated nucleic acid molecule over the converted
unmethylated molecule during the PCR.
[0044] In certain embodiments, a blocker sequence is added during
the PCR to minimize the amplification of the converted unmethylated
nucleic acid molecule.
[0045] The "blocker sequence" as used herein refers to a
polynucleotide, which is usually a synthetic polynucleotide that is
single-stranded and comprises a sequence that is hybridizable, and
preferably complementary, to a segment of the target nucleic acid
molecule, wherein the blocker sequence anneals to the target
nucleic acid molecule so as to block further primer extension of
the 3'-end of the first-strand cDNA at a desired position. Some
embodiments of strand displacement methods of the present invention
for obtaining a single-stranded DNA target sequence comprise use of
a blocker sequence. The blocker sequence comprises nucleotides that
bind to the target nucleic acid with an affinity, preferably a high
affinity, such that the blocker sequence resists displacement by
DNA polymerase in the course of primer extension, in preferably
more than about 30%, more preferably more than about 50%, even more
preferably more than about 75%, and most preferably more than about
90%, of primer extension events. The length and composition of the
blocker sequence should be such that excessive random non-specific
hybridization is avoided under the conditions of the methods of the
present invention. The length of the blocker polynucleotide is
preferably from about 3 to about 100 nucleotides, more preferably
from about 5 to about 80 nucleotides, even more preferably from
about 8 to about 40 nucleotides, and most preferably from about 10
to about 15 nucleotides. It is understood that the length can be
greater or less as appropriate under the reaction conditions of the
methods of this invention. The complementarity of the blocker
polynucleotide is preferably at least about 25%, more preferably at
least about 50%, even more preferably at least about 75%, and most
preferably at least about 90%, to its intended binding sequence on
the target nucleic acid. In some embodiments, the blocker sequence
that hybridizes to a DNA target nucleic acid is attached to the DNA
such that displacement of the blocker sequence by the polymerase
that affects primer extension is substantially, or at least
sufficiently, inhibited. Suitable methods for achieving such
attachment include, without limitation, techniques known in the
art, such as using a cytosine analog that contains a G-clamp
heterocycle modification as described by Flanagan et al., (Proc.
Natl. Acad. Sci. USA 96:3513-3518, 1999); and locked nucleic acids
as described, e.g., by Kumar et al., (Bioorg. Med. Chem. Lett.
8:2219-2222, 1998; and by Wahlestedt et al. (Proc. Natl. Acad. Sci.
USA 97:5633-5638, 2000), all of which are incorporated herein by
reference. Other suitable methods include using, where appropriate,
sequences with a high GC content and/or cross-linking. Any of these
methods for obtaining enhanced attachment may be used alone or in
combination. Alternatively, a molecule comprising a peptide nucleic
acid ("PNA") can be used. Blocker sequences are optional in the
methods of the present invention.
[0046] The PCR amplicons are further contacted with a
methylation-specific plasmonic nanoprobe comprising a plasmonic
nanoparticle and a non-ionic oligonucleotide analog probe
covalently coupled thereto, the oligonucleotide analog probe
comprising a base sequence that is complementary to the target
region, under conditions that allow the oligonucleotide analog
probe and the amplicons comprising the target region to hybridize
to each other.
[0047] Without wishing to be bound to any particular theory, the
probe in accordance with the present invention generates a
detectable signal if hybridized to the target nucleic acid molecule
that is distinguishable from the signal of the unhybridized probe.
Said signal may be any signal that is detectable by any means.
[0048] In preferred embodiments, these probes indicate the presence
or absence of the target by showing a color (e.g. red) in their
hybridized state and another color (e.g. light grey) in their
unhybridized, aggregated state. The aggregation of the unhybridized
nanoprobes may generally be achieved by control of the ionic
strength of the assay solution, for example by control of salt
concentrations. This particular behavior of the nanoprobes, i.e.
remaining in non-aggregated form as long as they are hybridized to
their target and aggregated if not hybridized to their target, can
be attributed to the non-ionic character of the nanoprobes.
[0049] The hybridization of the methylation-specific plasmonic
nanoprobe and the amplicons in step c) of the method may be carried
out at a temperature below the melting temperature of the duplex of
the nanoprobe and the amplicons of the target nucleic acid molecule
having the desired methylation status, i.e. typically the converted
methylated nucleic acid molecule, and above that of the duplex of
the nanoprobe and the amplicons of the target nucleic acid having
not the desired methylation status, i.e. typically the converted
unmethylated nucleic acid molecule, to allow maximum distinction
between these two groups. In these embodiments, step d) may also be
carried out at the above-described temperature by simply
determining the color of the assay solution that is indicative of
whether the hybrid has been formed or not. In these embodiments,
the melting temperature of the hybrid is thus only determined
insofar as it is determined whether the formed hybrid has a melting
temperature above the assay temperature, indicating the presence of
the target nucleic acid with the methylation status of interest, or
a melting temperature below the assay temperature, indicating the
absence of the target nucleic acid with the methylation status of
interest.
[0050] The term "nanoparticle" as used herein refers to any
particle having a size from about 1 to about 250 nm and has the
capacity to be covalently coupled to at least one oligonucleotide
analog as described herein. In certain embodiments, the
nanoparticle is a metal nanoparticle. In other embodiments, the
nanoparticle is a colloidal metal.
[0051] In some embodiments, the metal is a noble metal.
Non-limiting examples of a noble metal that can be used can include
silver, gold, platinum, palladium, ruthenium, osmium, iridium or
mixtures thereof, not to mention a few. Other metals that can also
be used in the formation of the nanoparticle can include but are
not limited to aluminium, copper, cobalt, indium, nickel, or any
other metal amenable to nanoparticle formation). The nanoparticle
as described herein can also comprise a semiconductor (including
for example and without limitation, CdSe, CdS, and CdS or CdSe
coated with ZnS) or magnetic (for example, ferromagnetite)
colloidal materials. Other nanoparticles useful in the practice of
the invention include, also without limitation, ZnS, ZnO, Ti,
TiO.sub.2, Sn, SnO.sub.2, Si, SiO.sub.2, Fe, Ag, Cu, Ni, Al, steel,
cobalt-chrome alloys, Cd, titanium alloys, AgI, AgBr, HgI.sub.2,
PbS, PbSe, ZnTe, CdTe, In.sub.2S.sub.3, In.sub.2Se.sub.3,
Cd.sub.3P.sub.2, Cd.sub.3As.sub.2, InAs, and GaAs.
[0052] The size of the nanoparticle used in the conjugate of the
present invention can vary in any size when desired, as long as the
nanoparticle is capable of providing optical properties; for
example, generate optical signals sensitive to hybridization
reactions. The diameter of the nanoparticle as described herein can
range in the size from about 1 nm to about 250 nm; about 1 nm to
about 200 nm; about 1 nm to about 160 nm; about 1 nm to about 140
nm; about 1 nm to about 120 nm; about 1 nm to about 80 nm; about 1
nm to about 60 nm; about 1 nm to about 50 nm; about 5 nm to about
250 nm; about 8 nm to about 250 nm; about 10 nm to about 250 nm;
about 20 nm to about 250 nm; about 30 nm to about 250 nm; about 40
nm to about 250 nm; about 85 nm to about 250 nm; about 100 nm to
about 250 nm; or about 150 nm to about 250 nm. In some embodiments,
the diameter of the diameter of the nanoparticle is in the range of
about 1 nm to about 100 nm.
[0053] In certain embodiments, the nanoparticle comprises a
surfactant. As used herein, "surfactant" refers to a surface active
agent which has both hydrophilic and hydrophobic parts in the
molecule. The surfactant can for example be used to stabilize the
nanoparticles. The surfactant can also be used to prevent
non-specific adsorption of the oligonucleotide analog on the
surface of the nanoparticles. In some embodiments, the surfactant
is a non-ionic surfactant. Other types of surfactants that can be
used can include but are not limited to cationic, anionic, or
zwitterionic surfactants. A particular surfactant may be used alone
or in combination with other surfactants. One class of surfactants
comprises a hydrophilic head group and a hydrophobic tail.
Hydrophilic head groups associated with anionic surfactants include
carboxylate, sulfonate, sulfate, phosphate, and phosphonate.
Hydrophilic head groups associated with cationic surfactants
include quaternary amine, sulfonium, and phosphonium. Quaternary
amines include quaternary ammonium, pyridinium, bipyridinium, and
imidazolium. Hydrophilic head groups associated with non-ionic
surfactants include alcohol and amide. Hydrophilic head groups
associated with zwitterionic surfactants include betaine. The
hydrophobic tail typically comprises a hydrocarbon chain. The
hydrocarbon chain typically comprises between about six and about
24 carbon atoms, more typically between about eight to about 16
carbon atoms.
[0054] The plasmonic nanoparticle for use in the present method is
functionalized with a non-ionic oligonucleotide analog probe that
preferably recognizes the amplicons of the converted methylated
nucleic acid molecule over those of the converted unmethylated
nucleic acid molecule. In certain embodiments, the oligonucleotide
analog probe is complementary to the amplicons at a region
comprising one or more CpG islands, and thus specifically
recognizes the amplicons of the converted methylated nucleic acid
molecule over those of the converted unmethylated molecule.
[0055] In some embodiments, the non-ionic oligonucleotide analog
probe used in the presently disclosed method is a morpholino
oligonucleotide probe or a derivative thereof. The term
"oligonucleotide analog" refers to an oligonucleotide having (i) a
modified backbone structure, e.g., a backbone other than the
standard phosphodiester linkage found in natural oligo- and
polynucleotides, and (ii) optionally, modified sugar moieties,
e.g., morpholino moieties rather than ribose or deoxyribose
moieties. The analog supports bases capable of hydrogen bonding by
Watson-Crick base pairing to standard polynucleotide bases, where
the analog backbone presents the bases in a manner to permit such
hydrogen bonding in a sequence-specific fashion between the
oligonucleotide analog molecule and bases in a standard
polynucleotide (e.g., single-stranded RNA or single-stranded DNA).
The analogs can for example, include those having a substantially
uncharged, phosphorus containing backbone.
[0056] A substantially uncharged, phosphorus containing backbone in
an oligonucleotide analog can for example be one in which a
majority of the subunit linkages, e.g., between 60-100%, are
uncharged at physiological pH, and contain a single phosphorous
atom. The oligonucleotide analog can comprise a nucleotide sequence
complementary to a target nucleic acid sequence as defined below.
In preferred embodiments, the oligonucleotide analogs of the
present invention are phosphorodiamidate morpholino oligos, wherein
the sugar and phosphate backbone is replaced by morpholine groups
linked by phosphoramidates and the nucleobases, such as cytosine,
guanine, adenine, thymine and uracil, are coupled to the morpholine
ring or derivatives thereof.
[0057] As used herein, the term "complementary" or
"complementarity" relates to the relationship of nucleotides/bases
on two different strands of DNA or RNA, or the relationship of
nucleotides/bases of the nucleotide sequence of the oligonucleotide
analog probe and a DNA/RNA strand, where the bases are paired (for
example by Watson-Crick base pairing: guanine with cytosine,
adenine with thymine (DNA) or uracil (RNA)). Therefore, the
oligonucleotide analog probe as described herein can comprise a
nucleotide sequence that can form hydrogen bond(s) with another
nucleotide sequence, for example a DNA or RNA sequence, by either
conventional Watson-Crick base pairing or other non-traditional
types of pairing such as Hoogsteen or reversed Hoogsteen hydrogen
bonding between complementary nucleosides or nucleotides. In this
context, the term "hybridize" or "hybridization" refers to an
interaction between two different strands of DNA or RNA or between
nucleotides/bases of the nucleotide sequence of the oligonucleotide
analog probe and a DNA/RNA sequence by hydrogen bonds in accordance
with the rules of Watson-Crick DNA complementarity, Hoogsteen
binding, or other sequence-specific binding known in the art. In
this context, it is understood in the art that a nucleotide
sequence of an oligonucleotide analog described herein need not be
100% complementary to a target nucleic acid sequence to be
specifically or selectively hybridizable. Complementarity is
indicated by a percentage of contiguous residues in a nucleic acid
molecule that can form hydrogen bonds with a second nucleic acid
molecule. For example, if a first nucleic acid molecule has 10
nucleotides and a second nucleic acid molecule has 10 nucleotides,
then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the
first and second nucleic acid molecules represents 50%, 60%, 70%,
80%, 90%, or 100% complementarity, respectively, not to mention a
few. Therefore, in some embodiments, the oligonucleotide analog
used herein can be 100% complementary to a target nucleic acid
molecule (i.e., a perfect match). In other embodiments, the
oligonucleotide analog probe can be at least about 95%
complementary, at least about 85% complementary, at least about 70%
complementary, at least about 65% complementary, at least about 55%
complementary, at least about 45% complementary, or at least about
30% complementary to the target nucleic acid molecule, provided
that it can specifically recognizes the amplicons of the converted
methylated nucleic acid molecule over those of the converted
unmethylated molecule.
[0058] The length of the oligonucleotide analog probe described
herein can comprise about 5 monomelic units to about 40 monomelic
units; about 10 monomelic units to about 35 monomelic units; or
about 15 monomelic units to about 35 monomelic units. The term
"monomeric unit" of an oligonucleotide analog probe as used herein
refers to one nucleotide unit of the oligonucleotide analog.
[0059] In certain embodiments, the oligonucleotide analog probe is
covalently coupled to the nanoparticle via a functional group. The
functional group is typically included in the spacer portion of the
oligonucleotide analog probe for covalently binding to the
nanoparticle. In some embodiments, the functional group can include
a thiol (SH) group, which can for example be used to covalently
attach to the surface of the nanoparticle. However, other
functional groups can also be used. Oligonucleotides functionalized
with thiols at their 3'-end or 5'-end can readily attach to gold
nanoparticles. See for example, Mucic et al. Chem. Commun. 555-557
(1996) which describes a method of attaching 3' thiol DNA to flat
gold surfaces. The thiol moiety also can be used to attach
oligonucleotides to other metal, semiconductor, and magnetic
colloids and to the other types of nanoparticles described herein.
Other functional groups for attaching oligonucleotides to solid
surfaces include phosphorothioate groups (see, for example, U.S.
Pat. No. 5,472,881 for the binding of
oligonucleotide-phosphorothioates to gold surfaces), substituted
alkylsiloxanes (see, for example Grabar et al., Anal. Ghent., 67,
735-743). Oligonucleotides having a 5' thionucleoside or a 3'
thionucleoside may also be used for attaching oligonucleotides to
solid surfaces. Other functional groups known to the skilled person
that can be used to attach the oligonucleotide analog probe to
nanoparticles can include but are not limited to disulfides such as
disulfide amides; carboxylic acids; aromatic ring compounds;
sulfolanes; sulfoxides; silanes, not to mention a few.
[0060] A more detailed description of the plasmonic nanoparticles
and nanoprobes for the practice of the present method may be found
in PCT international patent publication No. WO 2011/087456 A1,
which is hereby incorporated by reference in its entirety, with the
probe sequences adapted for the target of interest.
[0061] As set forth above, this method employs methylation-specific
PCR for the specific amplification of at least part of the
converted methylated nucleic acid molecule comprising a target
region the methylation status of which is to be analyzed, and
plasmonic nanoprobe-based colorimetric detection of the resultant
amplicons, and thus can be used to determine the methylation status
of the target nucleic acid molecule.
[0062] The plasmonic nanoprobes developed by the inventors of the
present invention are highly specific in recognition of nucleic
acid sequences. In preferred embodiments, plasmonic gold
nanoparticles are functionalized with non-ionic morpholino
oligonucleotides. Unlike the DNA-modified gold nanoparticles that
are stably dispersed in salt solution, the non-ionic nature of the
morpholino oligonucleotides makes the morpholino
oligonucleotides-modified nanoparticles much less stable, and only
dispersible in solutions with low ionic strength (e.g.,
[NaCl]<10 mmol/L). An increase of solution ionic strength would
lead to solution color change from red to light grey/colorless due
to nanoparticle aggregation. However, upon hybridization with
negatively charged DNA molecules, the nanoprobes become much more
stable due to the increase in surface charge, and the solution
remains red at a high ionic strength (e.g., [NaCl].about.100
mmol/L). When temperature rises, sharp melting transition occurs at
melting temperature (T.sub.m), whereby DNA molecules are released
from the nanoprobes, resulting in rapid color change in solution.
The nanoprobes are highly specific in recognizing DNA targets, and
a single-base mismatch may lead to the decrease in T.sub.m by
5-12.degree. C. This technology allows for accurate end-point
detection with standard equipment and a simple workflow. The
colorimetric signals can be easily visualized and recorded.
[0063] In certain embodiments, the method disclosed herein
comprises using a nucleic acid molecule comprising the unmethylated
target nucleotide sequence as a negative control, and/or using a
nucleic acid molecule comprising the methylated target nucleotide
sequence as a positive control.
[0064] The present method can be used to determine the methylation
status of the human Septin 9 (SEPT9) gene promoter, preferably in
the cell-free DNA (cfDNA) fragments circulating in the blood
released from colorectal cancer cells. In preferred embodiments,
the methylation-specific primers for use in the method have the
nucleic acid sequences 5'-ATTAGTTATTATGTCGGATTTCGC-3' (SEQ ID NO:
1) and 5'-CAACACGTCCGCGACCG-3' (SEQ ID NO: 2), the blocker has the
nucleic acid sequence 5'-GTTATTATGTTGGATTTTGTGGTTAATGTGTAG-3' and
is labelled with C3 spacer at the 3' end (SEQ ID NO: 3), and the
oligonucleotide analog probe used is a morpholino oligonucleotide
having the nucleic acid sequence 5'-CAACTACGCGTTAACCGCGATTTTT-3'
(SEQ ID NO: 4).
[0065] In another aspect, further disclosed herein is a kit for
determining the methylation status of a target nucleic acid
molecule in a sample, preferably of one or more CpG islands within
said target nucleic acid molecule, wherein the kit comprises a pair
of methylation-specific primers and a methylation-specific
plasmonic nanoprobe as described above.
[0066] In certain embodiments, the kit further comprises a blocker
sequence as described above.
[0067] In certain embodiments, the kit further comprises bisulfate
or a salt thereof.
[0068] In certain embodiments, the target nucleic acid molecule is
human SEPT9 gene, and the kit comprises a pair of
methylation-specific primers having the nucleic acid sequences
5'-ATTAGTTATTATGTCGGATTTCGC-3' (SEQ ID NO: 1) and
5'-CAACACGTCCGCGACCG-3' (SEQ ID NO: 2), and a plasmonic gold
nanoparticle functionalized with a morpholino oligonucleotide
having the nucleic acid sequence 5'-CAACTACGCGTTAACCGCGATTTTT-3'
(SEQ ID NO: 4). In preferred embodiments, the kit further comprises
a blocker having the nucleic acid sequence
5'-GTTATTATGTTGGATTTTGTGGTTAATGTGTAG-3' and is labelled with C3
spacer at the 3' end (SEQ ID NO: 3).
[0069] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
document, including definitions, will control.
[0070] The present invention is further illustrated by the
following examples. However, it should be understood, that the
invention is not limited to the exemplified embodiments.
EXAMPLES
Materials and Methods
A. Preparation of the Nanoprobes
[0071] The preparation of the nanoprobes used herein is similar to
that reported previously (Zu Y, et al. Anal Chem. 2011 Jun. 1;
83(11):4090-4; Zu Y, et al. Small. 2011 Feb. 7; 7(3):306-10).
Briefly, the MORs modified with disulfide amide at the 3' terminal
(Gene Tools, LLC) were treated with dithiothreitol, and then
purified by using an NAP-5 column (GE Healthcare). Gold NPs (40
nm-diameter, .about.0.1 nM) were mixed with .about.2 .mu.M of
thiolated MORs and 10 mM of phosphate buffer (pH 7.5), and allowed
to incubate at room temperature for 2 h. Next, the MOR-NP
conjugates were washed 5 times with a phosphate buffer solution (5
mM, pH 7.5) by centrifugation to remove the unreacted MORs. The
conjugates could be used immediately as nanoprobes or stored in
4.degree. C. refrigerator until use. The nanoprobes were stable for
at least 6 months when stored at 4.degree. C. Before use, the
nanoprobe solutions needed to be uniformly dispersed by
vortexing.
B. T.sub.m Measurements
[0072] The synthetic targets or PCR amplicons were simply mixed
with the specific nanoprobes. Next, the T.sub.m values of the
target-probe hybrids were measured with the thermal cycler. The
temperature was increased from 30.degree. C. at an interval of
1.0.degree. C. At each temperature, the solution was allowed to
incubate for 1 min prior to the color visualization or recording
with a camera. When a clear color change from red to light grey was
observed, the temperature was recorded as T.sub.m.
C. Standard gDNA Samples
[0073] Methylated and unmethylated gDNA samples purchased from
Promega were used as the standard gDNA template to evaluate the
assay performance.
D. Cell Line gDNA Extraction
[0074] Extraction of gDNA from cultured cells was performed with
the use of the Wizard.RTM. SV Genomic DNA Purification System
(Promega), according to the manufacturer's instructions. Quantity
(ng/.mu.l) and quality (A260/A280 ratio) of the gDNA samples were
checked by measuring the absorbance using Nanodrop 1000 (Thermo
Scientific). For SW480, RKO and LS174T gDNA samples, the A260/A280
ratios were 1.77, 1.82 and 1.81, respectively.
E. Bisulfite Conversion
[0075] Prior PCR amplification, gDNA samples were treated with
bisulfate to convert unmethylated cytosine to thymine. The
conversion was performed by using the MethylEdge Bisulfite
Conversion System (Promega), according to the manufacturer's
instructions.
F. aPCR
[0076] aPCR was used to produce single-stranded DNA targets. PCR
solution with a final volume of 25 .mu.L contained gDNA, 12.5 .mu.L
of master mix (Fermentas or Promega, 2.times.), 1000 nM of the
forward primer, 100 nM of the reverse primer, and 1000 nM of the
blocker. PCR cycling (Table 3) was performed on the PTC-200 DNA
Engine (Bio-Rad). The success of the PCR in producing specifically
sized amplicons was verified by running a 5-4 aliquot of the PCR
products on a 1.5% agarose gel stained with SafeView.TM. dye.
G. Determination of DNA Methylation
[0077] PCR products were mixed with the methylation-specific
nanoprobes. The solutions were allowed to incubate at 45.degree. C.
for 10 min. The solution color was then recorded by a digital
camera. Positive samples were identified by pink solutions, while
negative samples were identified by light grey/colorless
solutions.
TABLE-US-00001 TABLE 1 Sequences of primers, blocker and morpholino
probe used in this work. SEQ ID NO: Oligo Sequence (5' to 3') 1 PCR
forward ATT AGT TAT TAT GTC GGA TTT CGC primer 2 PCR reverse CAA
CAC GTC CGC GAC CG primer 3 Blocker GTT ATT ATG TTG GAT TTT GTG GTT
sequence AAT GTG TAG-C3 4 Morpholino CAA CTA CGC GTT AAC CGC GAT
TTT probe T
TABLE-US-00002 TABLE 2 Sequences of the synthetic DNA (representing
the target regions of methylated and un- methylated SEPT9 genes
after bisulfite conversion) used in this work. SEQ ID NO: Oligo
Sequence (5' to 3') 5 Synthetic GTT ATT ATG TCG GAT TTC GCG GTT
methylated AAC GCG TAG TTG GAT GGG ATT ATT DNA target TCG GAT TTC
GAA GGT GGG TGT TGG GTT GGT TGT TGC GGT CGC GGA CGT GT 6 Synthetic
GTT ATT ATG TTG GAT TTT GTG GTT unmethylated AAT GTG TAG TTG GAT
GGG ATT ATT DNA target TTG GAT TTT GAA GGT GGG TGT TGG GTT GGT TGT
TGT GGT TGT GGA TGT GT
TABLE-US-00003 TABLE 3 Thermal cycler protocol for PCR
amplification. Initial denaturing 95.degree. C. 3 min 70-cycle
amplification 95.degree. C. 20 sec 62.degree. C. 30 sec 72.degree.
C. 20 sec End 4.degree. C. Hold
H. Method
[0078] FIG. 1 shows the workflow of the test. Commercial kits were
used to obtain DNA and to perform bisulfite conversion, following
which, methylation-specific asymmetric PCR (aPCR) was conducted
using a standard thermal cycler. Next, PCR products were mixed with
nanoprobe assay solutions, and allowed to incubate at 45.degree. C.
for 10 min. Lastly, assay solution color was recorded by a digital
camera. Positive samples would display a pink color, while negative
samples would turn colorless.
[0079] As the concentration of circulating cfDNA in blood is very
low, and DNA damage may occur in the bisulfite conversion process,
the detection method must be highly sensitive. To increase
amplification yield, PCR cycle number was set at 70 cycles.
[0080] In the detection of methylated cfDNA, the interference of
normal non-methylated DNA in blood is usually significant. In
addition, bisulfite treatment could greatly reduce the level of
complexity in the DNA sequences, making the specific detection more
difficult. In the protocol, methylation-specific PCR primers that
involve at least 3 CpG sites were used. To inhibit the
amplification of unmethylated sequences, a blocker sequence was
also employed. The PCR products were gauged by a
methylation-specific nanoprobe that involves 4 CpG sites. The
sequences of PCR primers, blocker, and probe are shown in FIG. 2
and Table 1.
Example 1: Nanoprobe Evaluation
[0081] To characterize the nanoprobes, T.sub.m data were measured
in the presence of synthetic DNA samples over a broad concentration
range of 20 nM to 500 nM (FIG. 3). The synthetic DNA sequences
represent the target regions of methylated and unmethylated SEPT9
genes after bisulfite conversion (Table 2). At room temperature
(.about.25.degree. C.), the nanoprobes were stabilized by >20 nM
of the DNA samples. With the increase in DNA target concentration,
T.sub.m increases. The reduction of T.sub.m induced by 4 G/T
mismatches between the unmethylated target and the probe was
.about.15.degree. C.; this allowed for highly specific detection of
the methylated target.
Example 2: Assay Performance Evaluation
[0082] The sensitivity and specificity of the assay were tested by
using methylated and unmethylated human genomic DNA (gDNA) samples
with concentrations of 5 pM to 100 nM (corresponding copy numbers
are 1.5 and 3.times.10.sup.4, respectively). FIG. 4 shows that the
aPCR produced amplicons consistently for methylated gDNA down to 3
copies. Accordingly, the nanoprobe assay generated positive
results. For the samples with even lower concentrations, random
positive results were obtained.
[0083] Unmethylated gDNA was the interfering background of the
assay. The aPCR did not produce specific amplicons for the
unmethylated gDNA with concentration up to 50 nM. However, for
unmethylated gDNA of 100 nM, amplicon band appeared in the gel
electrophoresis analysis, indicating the non-specific amplification
of the unmethylated sequences. In this case, the specificity of the
methylation-specific primers and the effect of blockers were unable
to prohibit the aPCR from generating detectable amount of
unmethylated amplicons. Interestingly, in spite of the presence of
the non-specific products, the nanoprobe-based assay gave negative
results for the samples. The highly specific nanoprobes
successfully differentiated the unmethylated sequences from the
methylated ones.
[0084] In another test, the assay sensitivity in the presence of
100 nM of unmethylated gDNA was examined (FIG. 5). Similar to the
cases in the absence of unmethylated gDNA, the assay could detect 3
copies of methylated gDNA. Therefore, the assay is able to
consistently detect 0.01% of methylated gDNA in the background of
unmethylated sequences.
Example 3: Analysis of Cell Line Samples
[0085] gDNA extracted from several colorectal cancer cell line
samples, such as SW480, RKO and LS174T, was also tested. As
reported in the literature, these cell lines are all
hypermethylated in the promoter region of the SEPT9 gene. The assay
showed positive results in the methylation analysis for all the
samples (FIG. 6).
[0086] A highly sensitive and specific assay for SEPT9 gene
promoter methylation has been developed. The assay is able to
detect 0.01% methylated DNA in the background of unmethylated
sequences (i.e. .about.3 copies of methylated DNA in 100 nM of
unmethylated sequences). This assay would be suitable for
methylation analysis of cfDNA in blood samples.
[0087] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein. Other embodiments are within the
following claims. In addition, where features or aspects of the
invention are described in terms of Markush groups, those skilled
in the art will recognize that the invention is also thereby
described in terms of any individual member or subgroup of members
of the Markush group.
[0088] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. Further, it will be readily apparent to one skilled in the
art that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. The compositions, methods, procedures,
treatments, molecules and specific compounds described herein are
presently representative of preferred embodiments are exemplary and
are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention are
defined by the scope of the claims. The listing or discussion of a
previously published document in this specification should not
necessarily be taken as an acknowledgement that the document is
part of the state of the art or is common general knowledge.
[0089] The invention illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including," containing", etc.
shall be read expansively and without limitation. The word
"comprise" or variations such as "comprises" or "comprising" will
accordingly be understood to imply the inclusion of a stated
integer or groups of integers but not the exclusion of any other
integer or group of integers. Additionally, the terms and
expressions employed herein have been used as terms of description
and not of limitation, and there is no intention in the use of such
terms and expressions of excluding any equivalents of the features
shown and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by exemplary
embodiments and optional features, modification and variation of
the inventions embodied therein herein disclosed may be resorted to
by those skilled in the art, and that such modifications and
variations are considered to be within the scope of this
invention.
[0090] The content of all documents and patent documents cited
herein is incorporated by reference in their entirety.
Sequence CWU 1
1
9124DNAArtificialsynthetic construct 1attagttatt atgtcggatt tcgc
24217DNAArtificialsynthetic construct 2caacacgtcc gcgaccg
17333DNAArtificialsynthetic constructmisc_feature(33)..(33)n is G
labelled with C3 spacer 3gttattatgt tggattttgt ggttaatgtg tan
33425DNAArtificialsynthetic
constructmisc_feature(1)..(25)morpholino oligonucleotides
4caactacgcg ttaaccgcga ttttt 25598DNAArtificialsynthetic construct
5gttattatgt cggatttcgc ggttaacgcg tagttggatg ggattatttc ggatttcgaa
60ggtgggtgtt gggttggttg ttgcggtcgc ggacgtgt
98698DNAArtificialsynthetic construct 6gttattatgt tggattttgt
ggttaatgtg tagttggatg ggattatttt ggattttgaa 60ggtgggtgtt gggttggttg
ttgtggttgt ggatgtgt 987124DNAArtificialSept9 gene fragment
7gacccgctgc ccaccagcca tcatgtcgga ccccgcggtc aacgcgcagc tggatgggat
60catttcggac ttcgaaggtg ggtgctgggc tggctgctgc ggccgcggac gtgctggaga
120ggac 1248123DNAArtificialbisulfite treated hypermethylated Sept9
gene fragment 8gattcgttgt ttattagtta ttatgtcgga tttcgcggtt
aacgcgtagt tggatgggat 60tatttcggat ttcgaaggtg ggtgttgggt tggttgttgc
ggtcgcggac gtgttggaga 120gga 1239123DNAArtificialbisulfite treated
unmethylated Sept9 gene fragment 9gatttgttgt ttattagtta ttatgttgga
ttttgtggtt aatgtgtagt tggatgggat 60tattttggat tttgaaggtg ggtgttgggt
tggttgttgt ggttgtggat gtgttggaga 120gga 123
* * * * *