U.S. patent application number 11/252135 was filed with the patent office on 2006-04-27 for quantitative methylation detection in dna samples.
Invention is credited to Susan Cottrell.
Application Number | 20060088866 11/252135 |
Document ID | / |
Family ID | 27659063 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060088866 |
Kind Code |
A1 |
Cottrell; Susan |
April 27, 2006 |
Quantitative methylation detection in DNA samples
Abstract
Described is a method for methylation detection in a DNA sample.
An isolated genomic DNA sample is treated in a manner capable of
distinguishing methylated from unmethylated cytosine bases. The
pretreated DNA is amplified using at least one oligonucleotide
primer, a polymerase and a set of nucleotides of which at least one
is labeled with a first type of label. A sequence-specific
oligonucleotide probe, marked with a second type of label,
hybridizes to the amplification product and a FRET reaction occurs
if a labeled oligonucleotide is present in close proximity in the
amplification product. The method determines the level of
methylation of a sample by measuring the extent of fluorescence
resonance energy transfer (FRET) between the donor and acceptor
fluorophore.
Inventors: |
Cottrell; Susan; (Seattle,
WA) |
Correspondence
Address: |
KRIEGSMAN & KRIEGSMAN
665 Franklin Street
Framingham
MA
01702
US
|
Family ID: |
27659063 |
Appl. No.: |
11/252135 |
Filed: |
October 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10068553 |
Feb 6, 2002 |
6960436 |
|
|
11252135 |
Oct 17, 2005 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6827 20130101; C12Q 2535/113 20130101; C12Q 2545/114
20130101; C12Q 2531/107 20130101; C12Q 2535/113 20130101; C12Q
1/6827 20130101; C12Q 2531/107 20130101; C12Q 1/6818 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Claims
1. A method for the cytosine methylation detection in a DNA sample,
comprising the following steps: a) a genomic DNA sample is treated
in a manner capable of distinguishing methylated from unmethylated
cytosine bases; b) the pre-treated DNA is amplified using at least
one oligonucleotide primer, a polymerase and a set of nucleotides
of which at least one is marked with a first type of label; c) a
sequence-specific oligonucleotide or oligomer probe is hybridized
to the amplification product and a fluorescence resonance energy
transfer (FRET) occurs if the oligonucleotide or oligomer probe,
marked with a second type of label, binds in close proximity to one
of the labeled nucleotides that was incorporated into the
amplification product; d) the level of methylation of the sample is
determined by the level of interaction between said first and
second type of label.
2. A composition of pre-treated genomic DNA according to claim 1
for the determination of the methylation status of a corresponding
genomic DNA.
3. A diagnostic kit for the detection of the methylation of
cytosine bases in genomic DNA samples according to claim 1,
comprising reagents for the selective deamination of cytosine bases
in genomic DNA, one or more primers and labeled nucleotides for the
amplification step, a detectable probe and optionally protocols or
instructions for the method of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 10/068,553, filed Feb. 6, 2002, which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the analysis of nucleic acids,
especially to the analysis of methylation patterns in genomic DNA
by providing a means of detecting nucleotides, that are
characteristic for methylated sites after bisulfite treatment of
the genomic DNA. The method utilises the incorporation of labels
and the detection of fluorescence resonance energy transfer (FRET)
of the amplified sample DNA.
PRIOR ART
DNA Methylation
[0003] The levels of observation that have been studied in recent
years in molecular biology have concentrated on genes, the
translation of those genes into RNA, and the transcription of the
RNA into protein. There has been a more limited analysis of the
regulatory mechanisms associated with gene control. Gene
regulation, for example, at what stage of development of the
individual a gene is activated or inhibited, and the tissue
specific nature of this regulation is less understood. However, it
can be correlated with a high degree of probability to the extent
and nature of methylation of the gene or genome. From this
observation it is reasonable to infer that pathogenic genetic
disorders may be detected from irregular genetic methylation
patterns.
[0004] The efforts of the Human Genome project are concentrated on
the sequencing of the human genome. It is expected that this will
yield considerable therapeutic and diagnostic benefits for the
treatment of disease. However, these efforts have so far been
unable to address a significant aspect of genetic disorders, the
epigenetic factor. The epigenetic regulation of gene transcription
has been shown to effect many disorders. One of the most
significant epigenetic mechanisms so far identified has been the
methylation of cytosine. The methylation of cytosine at the 5
position is the only known modification of genomic DNA. Although
the exact mechanisms by which DNA methylation effects DNA
transcription are unknown, the relationship between disease and
methylation has been well documented. In particular methylation
patterns of CpG islands within regulatory regions of genome appear
to be highly tissue specific. Therefore, it follows that
misregulation of genes may be predicted by comparing their
methylation pattern with phenotypically `normal` expression
patterns. The following are cases of disease associated with
modified methylation patterns.
[0005] Head and neck cancer (Sanchez-Cespedes M et al. "Gene
promoter hypermethylation in tumors and serum of head and neck
cancer patients" Cancer Res. 2000 Feb. 15; 60 (4):892-5)
[0006] Hodgkin's disease (Garcia J F et al "Loss of p16 protein
expression associated wiht methylation of the p16INK4A gene is a
frequant finding in Hodgkin's disease" Lab invest 1999 December; 79
(12):1453-9)
[0007] Gastric cancer (Yanagisawa Y et al., "Methylation of the
hMLH1 promoter in familial gastric cancer with microsatellite
instability" Int J Cancer 2000 Jan. 1; 85 (1):50-3)
[0008] Prader-Willi/Angelman's syndrome (Zeschnigh et al "Imprinted
segments in the human genome: different DNA methylation patterns in
the Prader Willi/Angelman syndrome region as determined by the
genomic sequencing method" Human Mol. Genetics (1997) (6) 3 pp
387-395)
[0009] ICF syndrome (Tuck-Muller et al "CMDNA hypomethylation and
unusual chromosome instability in cell lines from ICF syndrome
patients" Cytogenet Call Genet 2000; 89(1-2):121-8
[0010] Dermatofibroma (Chen T C et al "Dermatofibroma is a clonal
proliferative disease" J Cutan Pathol 2000 January; 27
(1):36-9)
[0011] Hypertension (Lee S D et al., "Monoclonal endothelial cell
proliferation is present in primary but not secondary pulmonary
hypertension" J clin Invest 1998 Mar. 1, 101 (5):927-34)
[0012] Autism (Klauck S M et al., "Molecular genetic analysis of
the FMR-1 gene in a large collection of autistic patients" Human
Genet 1997 August; 100 (2): 224-9)
[0013] Fragile X Syndrome (Hornstra I K et al., "High resolution
methylation analysis of the FMR1 gene trinucleotide repeat region
in fragile X syndrome" Hum Mol Genet 1993 October,
2(10):1659-65)
[0014] Huntigton's disease (Ferluga J et al. "possible organ and
age related epigenetic factors in Huntington's disease and
colorectal carcinoma" Med hyptheses 1989 May; 29(1);51-4
[0015] All of the above documents are hereby incorporated by
reference.
Bisulphite Treatment
[0016] A relatively new and currently the most frequently used
method for analyzing DNA for 5-methylcytosine is based upon the
specific reaction of bisulfite with cytosine which, upon subsequent
alkaline hydrolysis, is converted to uracil which corresponds to
thymidine in its base pairing behaviour. However, 5-methylcytosine
remains unmodified under these conditions. Consequently, the
original DNA is converted in such a manner that methylcytosine,
which originally could not be distinguished from cytosine by its
hybridisation behaviour, can now be detected as the only remaining
cytosine using "normal" molecular biological techniques, for
example, by amplification and hybridisation or sequencing. All of
these techniques are based on base pairing which can now be fully
exploited. In terms of sensitivity, the prior art is defined by a
method which encloses the DNA to be analysed in an agarose matrix,
thus preventing the diffusion and renaturation of the DNA
(bisulfite only reacts with single-stranded DNA), and which
replaces all precipitation and purification steps with fast
dialysis (Olek A, Oswald J, Walter J. A modified and improved
method for bisulphite based cytosine methylation analysis. Nucleic
Acids Res. 1996 Dec. 15; 24(24):5064-6). Using this method, it is
possible to analyse individual cells, which illustrates the
potential of the method. However, currently only individual regions
of a length of up to approximately 3000 base pairs are analysed, a
global analysis of cells for thousands of possible methylation
events is not possible. However, this method cannot reliably
analyse very small fragments from small sample quantities either.
These are lost through the matrix in spite of the diffusion
protection.
[0017] An overview of the further known methods of detecting
5-methylcytosine may be gathered from the following review article:
Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998,
26, 2255.
[0018] To date, barring few exceptions (e.g., Zeschnigk M, Lich C,
Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the
diagnosis of Angelman and Prader-Willi syndrome based on allelic
methylation differences at the SNRPN locus. Eur J Hum Genet. 1997
March-April; 5(2):94-8) the bisulfite technique is only used in
research. Always, however, short, specific fragments of a known
gene are amplified subsequent to a bisulfite treatment and either
completely sequenced (Olek A, Walter J. The preimplantation
ontogeny of the H19 methylation imprint. Nat Genet. 1997 November;
17(3):275-6) or individual cytosine positions are detected by a
primer extension reaction (Gonzalgo M L, Jones P A. Rapid
quantitation of methylation differences at specific sites using
methylation-sensitive single nucleotide primer extension
(Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15; 25(12):2529-31, WO
Patent 9500669) or by enzymatic digestion (Xiong Z, Laird P W.
COBRA: a sensitive and quantitative DNA methylation assay. Nucleic
Acids Res. 1997 Jun. 15; 25(12):2532-4). In addition, detection by
hybridisation has also been described (Olek et al., WO 99
28498).
[0019] Further publications dealing with the use of the bisulfite
technique for methylation detection in individual genes are: Grigg
G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA.
Bioessays. 1994 June; 16(6):431-6, 431; zeschnigk M, Schmitz B,
Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments
in the human genome: different DNA methylation patterns in the
Prader-Willi/Angelman syndrome region as determined by the genomic
sequencing method. Hum Mol Genet. 1997 March; 6(3):387-95; Feil R,
Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on
individual chromosomes: improved protocol for bisulphite genomic
sequencing. Nucleic Acids Res. 1994 Feb. 25; 22(4):695-6; Martin V,
Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing
indicates a correlation between DNA hypomethylation in the 51
region of the pS2 gene and its expression in human breast cancer
cell lines. Gene. 1995 May 19; 157(1-2):261-4; WO 97 46705, WO 95
15373 and WO 45560.
Fluorescence Resonance Energy Transfer (FRET)
[0020] Fluorescence resonance energy transfer (FRET) is an
interaction between two molecules wherein the excited state of one
molecule (the donor) transfers energy to the other molecule (the
acceptor). The donor molecule is a fluorophore while the acceptor
molecule may or may not be. The energy transfer occurs without the
emission of photons, and is based on dipole-dipole interactions
between the two molecules. Molecules that are commonly used in FRET
include fluorescein, N,N,N',N'-tetramethyl-6-carboxyrhodamine
(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)
benzoic acid (DABCYL), and
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).
[0021] Basic conditions for FRET include the following:
[0022] Close proximity between the donor and acceptor molecules
(typically 10-100 .ANG.).
[0023] The emission spectrum of the donor molecule must overlap the
absorption spectrum of the acceptor molecule The transition dipole
orientations of the donor and acceptor molecules must be
approximately parallel.
[0024] The extent of the energy transfer is dependent on the
distance between the two molecules, and the overlap between the
donor and acceptor spectra. It can be described by the following
equation: kt(r)=tD-1*(R0/r)6 wherein r is the distance between the
donor and the acceptor tD is the lifetime of the donor in the
absence of energy transfer R0 is termed the Forster distance.
[0025] The efficiency of the energy transfer (for a single
donor-acceptor pair) is given by: E=R06/(R06+r6)
[0026] Forster distances are typically in the range of 30-60 .ANG..
Therefore FRET can be used as a highly sensitive method of
measuring microscopic distances, this is particularly useful within
the field of molecular biology where it has been utilised in a
number of ways. It has been used in the study of protein structure,
assembly, distribution, conformation and interactions, as well as
the study of cell membranes and immunoassays. FRET has also been
used in a number of ways in the analysis of nucleic acids. This
includes the analysis of the structure and conformation of nucleic
acids, hybridisation, PCR, sequencing and primer extension
assays.
[0027] Another class of probe labels include fluorescence
quenchers. The emission spectra of a quencher overlaps with a,
fluorescent dye such that the fluorescence of the fluorescent dye
is substantially diminished, or quenched, by the phenomena of
fluorescence resonance energy transfer "FRET" (Clegg (1992) Meth.
Enzymol., 211:353-388). A fluorescent reporter dye and quencher
joined in a configuration that permits energy transfer from the
fluorophore to the quencher may result in a reduction of the
fluorescence of the fluorescent dye. The reporter is a luminescent
compound that can be excited either by chemical reaction, producing
chemiluminescence, or by light adsorption, producing fluorescence.
The quencher can interact with the reporter to alter its light
emission, usually resulting in the decreased emission efficiency of
the reporter. The efficiency of this quenching phenomenon is
directly correlated with the distance between the reporter molecule
and the quencher molecule (Yaron (1979) Analytical Biochemistry,
95:228-35).
[0028] Particular quenchers include but are not limited to
rhodamine dyes such as tetramethyl-6-carboxyrhodamine (TAMRA) or
tetrapropano-6-carboxyrhodamine (ROX) (Bergot, U.S. Pat. No.
5,366,860).
Enzymatic Amplification
[0029] PCR is a commonly used technique that has been described,
for example in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159.
Briefly, it is the amplification of a nucleic acid sequence by
repetitive cycles of annealing and extending primer to single
stranded nucleic acids followed by the denaturation of the
resultant double stranded molecule. PCR, (and variations thereof)
has a multitude of applications and is one of the key technologies
involved in most forms of nucleic acid analysis and
manipulation.
[0030] An important variation is the Multiplex-PCR, where more then
2 specific primer are used and a multitude of different specific
amplificats are obtained in one reaction chamber.
[0031] There are several commonly used methods for the detection of
PCR products, such as gel electrophoresis and the use of labelled
primer oligonucleotides and nucleoside triphosphates. The use of
fluorescent labelled nucleotides and oligomers within PCR for
nucleic acid analysis is also known.
PNA FRET Probes
[0032] PNA can hybridise to its target complement in either a
parallel or anti-parallel orientation. However, the anti-parallel
duplex (where the carboxyl terminus of PNA is aligned with the
5'terminus of DNA, and the amino terminus is aligned with the
3'-terminus of DNA) is typically more stable (Egholm (1993) Nature,
365:566-68). PNA probes are known to bind to target DNA sequences
with high specificity and affinity (Coull, U.S. Pat. No.
6,110,676). The PNA FRET probe examples of the present invention,
with reporter or quencher moieties, are designed such that the PNA
anneals in the anti-parallel orientation with the target
sequences.
[0033] PNA may be synthesized at any scale on automated
synthesizers. The PNA FRET probes may be synthesized on many of the
commonly used solid supports. After synthesis is complete, the PNA
may be cleaved from the support, purified, analysed and
quantitated. Fluorescent-labeled PNA probes have demonstrated
desirable properties in hybridization assays (Hyldig-Nielsen, U.S.
Pat. No. 5,985,563).
[0034] Genomic DNA for further amplification is obtained from DNA
of cells, tissue or other test samples using standard methods. This
standard methodology is found in references such as Fritsch and
Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.
Real Time PCR
[0035] Real time PCR monitoring using fluorescence has been
described in several manners. Firstly, the binding of double
stranded DNA specific fluorescent dyes such as ethidium bromide
allows for the monitoring of the accumulation of PCR product by
correlation with increased fluorescence. A second detection method,
polymerase mediated exonuclease cleavage utilises the 5'
exonuclease activity of polymerases such as Taq. An oligonucleotide
probe that is complementary to the PCR product, yet distinct from
the PCR primer is labelled with a FRET pair such that the donor
molecule is quenched by an acceptor molecule. During PCR
amplification, the 5' exonuclease proceeds to digest the probe,
separating the FRET pair and leading to increased fluorescence. A
variation on this technology uses a nucleic acid wherein the FRET
pair is internally quenched, for example, by having a hairpin
conformation. Upon hybridisation to a sequence of interest, the
FRET pair is separated and the donor molecule emits fluorescence.
This technology can be used, for example for the analysis of
SNPs.
[0036] An alternative technology is based on the use of two species
of hybridisation probes, each labelled with a member of a FRET
pair. Upon hybridisation of both probes to the target sequence in
adequate proximity, a fluorescent signal is emitted. Again, this
technology may be used for the detection of SNPs.
[0037] A major advantage of the use of such FRET based PCR
technologies is that the reaction may be monitored in a closed tube
reaction, suitable for use in high and medium throughput and
reducing the probability of contamination.
DESCRIPTION OF THE INVENTION
[0038] According to the present invention there is provided a
method for the cytosine methylation detection in a DNA sample,
comprising the following steps:
a) a genomic DNA sample is treated in a manner capable of
distinguishing methylated from unmethylated cytosine bases;
b) the pre-treated DNA is amplified using at least one
oligonucleotide primer, a polymerase and a set of nucleotides of
which at least one is marked with a first type of label;
[0039] c) a sequence-specific oligonucleotide or oligomer probe is
hybridized to the amplification product and a FRET occurs if the
oligonucleotide or oligomer probe, marked with a second type of
label, binds in close proximity to one of the labeled nucleotides
that was incorporated into the amplification product;
d) the level of methylation of the sample is determined by the
level of interaction between said first and second type of
label.
[0040] According to the invention it is preferred that the first
type of label is a donor fluorophore and the second type of label
is an acceptor fluorophore and that the extent of fluorescence
resonance energy transfer (FRET) is measured. It is further
preferred that the first type of label is an acceptor fluorophore
and the second type of label is a donor fluorophore and that the
extent of fluorescence resonance energy transfer (FRET) is
measured.
[0041] A further preferred embodiment of the present invention is
characterised in that the nucleotides of step b) contain a
fluorescent moiety and the probe in step c) a quencher moiety. It
is also preferred according to the invention that the nucleotides
of step b) contain a quencher moiety and the probe in step c) a
fluorescent moiety.
[0042] According to the invention it is also preferred that the
polymerase has no 5' to 3' exonuclease activity in order to prevent
degradation of the probe.
[0043] It is further preferred according to the invention that a
change in fluorescence intensity is monitored in real-time during
the amplification reaction.
[0044] It is also especially preferred according to the present
invention that a change in fluorescence intensity is monitored at
end-point of target amplification.
[0045] According to another preferred embodiment of the present
invention the amplification reaction is achieved with the
polymerase chain reaction (PCR).
[0046] According to the invention it is preferred that the probe
contains only one CpG or that the probe contains several CpGs.
Especially in this case it is further preferred that each probe for
each CpG has a fluorescent label.
[0047] In a further preferred embodiment of the present invention
the probe can be end labeled or internally labeled.
[0048] It is also preferred according to the invention that the
methylation information is determined by the change in fluorescence
intensity during subsequent rounds or cycles of PCR.
[0049] It is also preferred that the sample DNA is only amplified
by chosen PCR primers if a certain methylation state is present at
a specific site in the sample DNA.
[0050] According to the present invention a method is preferred
wherein the sample DNA is only amplified if a certain methylation
state was present at a specific site in the sample DNA, the
sequence context of which is essentially complementary to one or
more oligonucleotides or PNA oligomers which are additionally used
in the PCR reaction.
[0051] It is also preferred that the amplification from the 3'-end
of the probe is blocked by phosphorylation.
[0052] According to the invention it is also preferred that a
melting curve is generated at the end of the PCR to gather
additional data.
[0053] It is especially preferred within the scope of the present
invention that the fluorescent moiety is a fluorescein dye, a
rhodamine dye, or a cyanine dye. Especially preferred is also that
the quencher moiety is a rhodamine dye.
[0054] It is an especially preferred feature of the present
invention that the deamination treatment of the DNA is performed
with a bisulfite reagent.
[0055] It is also preferred according to the invention that the DNA
sample is cleaved prior to deamination treatment with restriction
endonucleases.
[0056] In a preferred embodiment of the method of the invention the
DNA sample is isolated from mammalian sources e.g. cell lines,
blood, sputum, faeces, urine, cerebrospinal fluid, tissue embedded
in paraffin, for example, ocular tissue, intestine, kidney, brain,
heart, prostate, lung, chest or liver, histological slides and all
possible combinations.
[0057] It is another preferred embodiment of the present invention
to use of a pre-treated genomic DNA within the method according to
the present invention for the determination of the methylation
status of a corresponding genomic DNA.
[0058] Another object of the present invention is to provide a
diagnostic kit for the detection of the methylation of cytosine
bases in genomic DNA samples, comprising reagents for the selective
deamination of cytosine bases in genomic DNA, one or more primers
and labeled nucleotides for the amplification step, a detectable
probe and optionally protocols or instructions for one of the
methods according to one of the preceding claims.
[0059] The invention describes a method to determine the presence
of specific CpG dinucleotides in a fragment of DNA using
fluorescence resonance energy transfer (FRET). This can be used to
obtain information about sequence properties of a sample DNA
fragment. For example, a point mutation could be identified in a
fragment if a nucleotide is present in its sequence as a result of
this mutation which is not present in the wild type.
[0060] The method is preferably used to measure cytosine
methylation. As mentioned above, bisulphite leads to the selective
deamination of cytosine, leaving 5-methylcytosine essentially
unchanged. Methylation of cytosine occurs almost exclusively in the
sequence context 5'-CG-3'. Therefore, after bisulphite treatment,
certain dinucleotides containing C do not occur anymore in one
strand, but they may still occur in the complementary strand formed
in the amplification of bisulphite treated DNA, for example using
the polymerase chain reaction (PCR).
[0061] This invention provides a method for visualizing the
methylation status of a CpG at defined positions in a very
sensitive way with very low background signal.
[0062] The method briefly comprises the following steps of
a) treating a genomic DNA sample in a manner capable of
distinguishing methylated from unmethylated cytosine bases;
b) amplifying the pre-treated DNA using at least one
oligonucleotide primer, a polymerase and a set of nucleotides of
which at least one is marked with a first type of label;
[0063] c) hybridising a sequence-specific oligonucleotide or
oligomer probe to the amplification product, a FRET occurs if the
oligonucleotide or oligomer probe, marked with a second type of
label, binds in close proximity to one of the labeled nucleotides
that was incorporated into the amplification product;
d) determining the level of methylation of the sample by measuring
by the level of interaction between said first and second type of
label.
[0064] In a preferred embodiment of the invention, the first type
of label is a donor fluorophore and the second type of label is an
acceptor fluorophore and that the extent of fluorescence resonance
energy transfer (FRET) is measured.
[0065] In a further preferred embodiment of the invention, said
first type of label is an acceptor fluorophore and the second type
of label is a donor fluorophore and that the extent of fluorescence
resonance energy transfer (FRET) is measured.
[0066] It is preferred that two or more CpGs are separately
interrogated in an amplification reaction.
[0067] Preferably, separate probes for each CpG are used, each with
its own fluorescent label.
[0068] The instant invention also enables a multiplex PCR to
rapidly determine optimal assay parameters and a fast,
cost-effective, and accurate system for the quantitative analysis
of target analytes. A multiplexed assay can for example be designed
in a standard 96 well microtiter plate format at room temperature
using conventional robotic systems for sample delivery and
preparation.
[0069] Preferably, the oligonucleotide or oligomer probes used
comprise one or more nucleotide analogs selected from a nucleobase
analog, a 2'-deoxyribose analog, an internucleotide analog, PNA or
LNA.
[0070] Although the detection of C or G nucleotides (in the
complementary strand) after bisulphite treatment in the context CpG
is the preferred application, also any other nucleotide or
polymorphism can in principle be detected. The preferred
application is to determine the methylation status of certain CpG
positions by determining the level of interaction between an
unconverted C in the bisulphite treated DNA and a labeled
oligonucleotide hybridised thereto. Accordingly, Guanin in the
complementary strand (after PCR) can be used for the same
purpose.
[0071] Also, converted positions after bisulphite treatment can be
identified by detecting thymine (or adenine in the complementary
strand) at selected positions using this technology. However,
design of probes becomes more difficult as it is not possible to
distinguish between T that was in the original genomic sequence and
T positions that were created through bisulphite conversion,
indicating lack of methylation at the respective cytosines.
[0072] In more detail, this method for the detection of specific
nucleotides in a DNA sample is characterized in that an isolated
genomic DNA sample is treated in a manner capable of distinguishing
methylated from unmethylated cytosine bases and the pre-treated DNA
is amplified using at least one oligonucleotide primer, a
polymerase and a set of nucleotides at least one of which is marked
with a first type of label, in a first embodiment a donor
fluorophore and in a second embodiment an acceptor fluorophore.
[0073] Preferably, the acceptor and donor dyes (fluorophores) are
chosen in a way that the emission wavelength of the donor dye
overlaps with the excitation wavelength of the acceptor dye. It is
preferred that the emission and excitation spectra are sharp peaks
and that the emission spectra of the dyes are unlikely to
overlap.
[0074] It is preferred that the polymerase has no 5' to 3'
exonuclease activity to prevent degradation of the probe.
[0075] For example, dGTP is labeled with a fluorescent dye. The
labeled dGTP is incorporated only where there was a methylated
cytosine in the original DNA sample. Alternatively, the dCTP can be
labeled with a fluorescent dye.
[0076] After the extension phase, the DNA is denaturated and then
allowed to reanneal.
[0077] A sequence-specific oligonucleotide or oligomer probe
(referred to as oligomer probes if DNA analogs like PNA are used),
marked with a second type of label that is in a first embodiment an
acceptor fluorophore and in a second embodiment a donor fluorophore
hybridizes to the amplification product. Preferably, amplification
from the 3' end of the probe is blocked by phosphorylation (with
didesoxynucleotides).
[0078] The labels are preferably introduced into the
oligonucleotide probes by standard enzymatic methods, such as the
use of 5' labeled amplification primers for 5' labeling or
fluorescent-labeled base analogs for internal labeling.
[0079] A FRET reaction occurs if the fluorescently labeled
oligonucleotide, preferably in one embodiment a fluorescent moiety
binds in close proximity to a nucleotide labeled with a quencher
moiety, that was incorporated into the amplification product or in
another embodiment a quencher moiety that binds in close proximity
to a nucleotide labeled with a fluorescent moiety that was
incorporated into the amplification product (FIG. 1).
[0080] It is preferred that the probe is positioned in several
places relative to the CpG. The conformation of the DNA controls
the spacing of the two fluorescent dyes. Preferently, the
positioning is optimized for each CpG.
[0081] In a preferred embodiment, the probe is separated from the
amplification primers or alternatively it is attached to the 5' end
of one of the primers.
[0082] This way, the level of methylation can be determined,
identifying the CG dinucleotides. If a TG dinucleotide is present
instead, no FRET will be observed. Therefore, this method can be
directly used to monitor DNA methylation (FIG. 2).
[0083] In a preferred embodiment of the invention a real time
monitoring of the FRET signal is performed during the amplification
reaction. This way, the progress of the amplification can be
examined (FIG. 3). Very preferably the amplification reaction is a
polymerase chain reaction (PCR), even though other amplification
procedures for example cloning or SDA (Strand Displacement
Amplification) are also preferred.
[0084] In another preferred embodiment of the invention the change
in fluorescence intensity is monitored at end-point of target
amplification. End-point analysis of the PCR entails fluorescent
dye signal measurement when thermal cycling and amplification is
complete. Results are reported in terms of the change in
fluorescence, i.e. fluorescence intensity units, of the fluorescent
dye signal from start to finish of the PCR thermal cycling,
preferably minus any internal control signals.
[0085] It is also preferred that a melting curve is generated at
the end of the PCR to gather additional data.
[0086] In another preferred embodiment of the invention the CpG
dinucleotide occurs only once in the amplification product. As
outlined above, this is very helpful if the presence of the FRET
signal is directly used to draw conclusions about the sequence
characteristics of the sample DNA.
[0087] For example, if only one labeled CG is present in an
amplification product of a bisulphite treated sample and a
fluorescently labeled probe binds in close proximity to it, a FRET
occurs and direct conclusions can be drawn that a methylated
cytosine was present in a certain position in the genomic DNA
sample.
[0088] If several labeled CGs are present for example in an
amplification product of a bisulphite treated sample and probes
bind in close proximity to them, each with its own fluorescent
label, several FRET reactions occur and conclusions can be drawn
about the methylated cytosines from all sites involved.
[0089] In a further preferred embodiment of the invention the
methylation information is determined by the change in fluorescence
intensity during subsequent rounds of PCR.
[0090] Preferably, the sample is illuminated during the
amplification reaction with light of appropriate wavelength.
[0091] In a preferred embodiment of the invention, prior to the PCR
either essentially all cytosines in the DNA sample are selectively
deaminated, but 5-methylcytosines remain essentially unchanged or
essentially all 5-methylcytosines in the DNA sample are selectively
deaminated, but cytosines remain essentially unchanged.
Cytosine-guanine (CpG) dinucleotides are detected, allowing
conclusions about the methylation state of cytosines in said CpG
dinucleotides in said DNA sample. This deamination is preferably
performed using a bisulphite reagent.
[0092] Preferably, the sample DNA is only amplified by chosen PCR
primers if a certain methylation state is present at a specific
site in the sample DNA the sequence context of which is essentially
complementary to one or more of said chosen PCR primers. This can
be done using primers annealing selectively to bisulphite treated
DNA which contains in a certain position either a TG or a CG,
depending on the methylation status in the genomic DNA. Primers can
be designed for both cases. A primer could contain a G at its
3'-end, therefore if would only bind to a DNA containing a C at the
respective position and therefore this primer will only or
preferentially amplify methylated DNA because the C is indicative
of a methylation in this position after bisulphite treatment. This
method is known as MSP, methylation sensitive PCR.
[0093] In another preferred embodiment of the invention, the sample
DNA is only amplified if a certain methylation state is present at
a specific site in the sample DNA the sequence context of which is
essentially complementary to one or more oligonucleotides or PNA
oligomers which are additionally used in the PCR reaction. These
oligonucleotides or PNA oligomers bind selectively to the template
DNA and prevent its amplification depending on the methylation
state of the DNA prior to bisulphite conversion.
[0094] Preferably, the fluorescent moiety is a fluorescein dye, a
rhodamine dye, or a cyanine dye and the quencher moiety a rhodamine
dye.
[0095] In another preferred variant of the invention the DNA sample
is cleaved prior to deamination (for example bisulphite) treatment
with restriction endonucleases.
[0096] Preferred is also a method whereby the enzymatic
amplification of the treated DNA is such that only one strand of
the DNA sample is amplified.
[0097] Preferably, the DNA sample is isolated from mammalian
sources e.g. cell lines, blood, sputum, faeces, urine,
cerebrospinal fluid, tissue embedded in paraffin, for example,
ocular tissue, intestine, kidney, brain, heart, prostate, lung,
chest or liver, histological slides and all possible
combinations.
[0098] Another embodiment of the present invention is a diagnostic
kit for the detection of the methylation of cytosine bases in
genomic DNA samples, comprising reagents for the selective
deamination of cytosine bases in genomic DNA, one or more primers
and fluorescently labeled nucleotides for the amplification step
and optionally protocols or instructions for one of the methods
according to one of the preceding claims.
[0099] This kit can also comprise several additional items for
example detectable probes.
[0100] The components of said kit, as an example, could comprise
receptacles for the following in sufficient quantities to carry out
the method: [0101] 1) Reagents for the bisulfite conversion of
sample DNA. [0102] 2) Reagents for the amplification of the
converted sample and incorporation of fluorophore labelled
nucleotides including: [0103] a) nucleic acid primer and [0104] b)
appropriate mix of nonlabeled and fluorophore labeled nucleotides
and [0105] c) DNA polymerase able to incorporate the fluorophore
labelled nucleotides [0106] 3) Instructions for use
[0107] The term `instructions for use` should cover a tangible
expression describing the reagent concentrations for the assay
method, parameters such as the relative amounts of reagents to be
combined, maintenance times for reagents/sample mixtures,
temperature, buffer conditions and the like.
[0108] In the following, steps of preferred embodiments of the
invention are described in more detail.
DNA Isolation
[0109] The genomic DNA sample must be isolated from tissue or
cellular sources. For mammals, more preferably humans, the DNA
sample may be taken from any tissue suspected of expressing the
target site within the genome and also from, such as cell lines,
blood, sputum, faeces, urine, cerebrospinal fluid, tissue embedded
in paraffin; for example, tissue of intestine, kidney, brain,
heart, prostate, lung, chest or liver, histological slides, but not
limited to those. Extraction may be by means that are standard to
one skilled in the art, these include the use of detergent lysates,
sonification and vortexing with glass beads. Once the nucleic acids
have been extracted the genomic double stranded DNA is used for
analysis.
Bisulfite Treatment
[0110] The sample DNA is then treated chemically in order to
convert the unmethylated cytosine bases into uracil. The chemical
modification may be by means of, for example, (but not limited to)
a bisulfite solution. Said chemical conversion may take place in
any format standard in the the art. This includes but is not
limited to modification within agarose gel or in denaturing
solvents.
[0111] Wherein the chemical modification takes the form of a
bisulfite treatment of the DNA the following steps may be
followed.
[0112] The double stranded DNA must be denatured. This may take the
form of a heat denaturation carried out at variable temperatures.
For high molecular weight DNA, the denaturation temperature is
generally greater than 90.degree. C. However, the analysis may be
upon smaller fragments which do not require such high temperatures.
In addition as the reaction proceeds and the cytosine residues are
converted to uracil the complementarity between the strands
decreases. Therefore, a cyclic reaction protocol may consist of
variable denaturation temperatures.
[0113] The bisulfite conversion then consists of two important
steps, the sulfonation of the cytosine and the subsequent
deamination. The equilibra of the reaction are on the correct side
at two different temperatures for each stage of the reaction.
Taking into account the kinetics of the reactions it is preferable
that the reaction takes place under cyclic conditions, with
changing temperatures. The temperatures and length at which each
stage is carried out may be varied according to the specific
requirement of the situation. However, a preferred variant of the
method comprises a change of temperature from 4 C (10 minutes) to
50 C (20 minutes). This form of bisulfite treatment is state of the
art with reference to WO 99/28498.
[0114] Said chemical conversion may take place in any format
standard in the art. This includes but is not limited to
modification within agarose gel, in denaturing solvents or within
capillaries.
[0115] Bisulfite conversion within agarose gel is state of the art
and has been described by Olek et al, Nucl. Acids. Res. 1996, 24,
5064-5066. The DNA fragment is embedded in agarose gel and the
conversion of cytosine to uracil takes place with hydrogensulfite
and a radical scavenger. The DNA may then be amplified without need
for further purification steps.
[0116] In a further preferred embodiment the DNA conversion may
take place without an agarose matrix. The DNA may incubated at
increased temperatures with hydrogensulfite and a radical
scavenger. Said reaction takes place within an organic denaturing
solvent. Examples of denaturing solvents include, but are not
limited to, Polyethylene glycol dialkyl
polyethylenglycol-dialkylether, dioxane and substituted
derivatives, urea or derivatives, acetonitrile, primary alcohols,
secondary alcohols, tertiary alcohols, DMSO or THF.
[0117] In a further embodiment, prior to chemical treatment the DNA
sample is transferred into a heatable capillary that is permeable
to small molecules. The reaction steps of the chemical modification
may then be carried out in the capillary tubes by means of the
addition and removal of reagents through connected capillaries.
[0118] Subsequent to the chemical treatment the two strands of the
DNA may no longer be complementary.
[0119] Amplification and Incorporation of labeled Nucleotides
Fractions of the so treated genomic DNA are then enzymatically
amplified using oligonucleotide primers. The length and design of
said primers may be specific to the area of the genome to be
analysed. As such a wide range of primers are suitable for use in
this technique. Such primer design is within the state of the
art.
[0120] An appropriate fraction of the nucleotides presented in the
amplification reaction, for example the G nucleotides, are labeled
with either a first or second type of label, the first type being a
fluorophore and the second type a quencher or vice versa.
Acceptable fluorophores for labeling the nucleotides are well known
to those skilled in the art and include, but are not limited to,
fluorescein, rhodamine, cyanine, phycoerythrin, Cy 5, Cy 5.5, Cy 7,
LC Red 640 or LC Red 705 whereas the acceptable quenchers are
rhodamine dyes. Attaching those dyes to the nucleotides lies within
the state of the art.
[0121] In a preferred embodiment of this invention the sample is
illuminated during the amplification reaction with light of
appropriate wavelength.
[0122] The skill of the invention lies in the interpretation of a
FRET signal during states, where a sequence-specific
oligonucleotide probe is hybridized to an amplification product and
a FRET occurs if the fluorescently labeled oligonucleotide binds in
close proximity to one of the labeled nucleotides that were
incorporated into the amplification product in order to gain
knowledge of the methylation state of the sample.
Advanced Data Processing
[0123] It is anticipated that the method will be used for the high
throughput analysis of genomic DNA samples. Therefore, the
invention also involves analysis of data using a computing device.
In a preferred embodiment said device may comprise one or more
databases. In a further preferred embodiment said device may
comprise one or more learning algorithms.
DESCRIPTION OF THE DRAWINGS
[0124] FIG. 1
Legend:
[0125] 1: Bisulfite Treatment [0126] 2: Incorporation of labeled
nucleotide [0127] 3: Detection of target by Fret [0128] 4:
Real-Time PCR
[0129] DNA extracted from a tissue is treated with sodium bisulfite
(A). Into the bisulfite treated DNA (single strand (B) dGTPs are
incorporated (C+D) that are labeled with a fluorescent dye during a
real-time PCR. A FRET occurs if the fluorescently labeled
oligonucleotide binds in close proximity to one of the labeled
nucleotides that was incorporated into the amplification product
(E).
[0130] FIG. 2
Legend:
[0131] 1. Bisulfite treatment [0132] 2. PCR with labeled dGTP
[0133] 3. Extension of primer [0134] 4. Denaturation [0135] 5.
Annealing and fluorescence monitoring [0136] 6. More Cycles
[0137] In the first step DNA of interest (SEQ ID NO:1) is
chemically treated to yield the sequence shown in the second row
(SEQ ID NO:2), wherein the only cytosines remaining in the sequence
are those that were methylated in the original sample. PCR primers
(shown in the third row, SEQ ID NO:3) designed to target one of the
DNA strands anneal to the template (SEQ ID NO:2) and extend it by
incorporating labeled nucleotides (dGTP) to yield SEQ ID NO:4. The
labeled dGTP is incorporated only where there was a methylated
cytosine in the original DNA sample. After the extension phase, the
DNA (SEQ ID NO:2, sixth row) is denaturated, allowed to reanneal
with primers (SEQ ID NO:5, left, and SEQ ID NO:6, right) and the
fluorescence monitored. With each round of PCR, more targets
complementary to the probe accumulate. The amount of fluorescence
emitted from the probe is measured.
[0138] FIG. 3
Legend:
[0139] 1. PCR with unlabeled dCTP, dATP, dTTP and labeled dGTP
[0140] 2. Hybridization of fluorescent-labeled gene specific
oligo-nucleotide [0141] 3. FRET, real-time fluorescence
detection
[0142] A PCR is performed with primers that target one of the DNA
strands (SEQ ID NO:7, first row, and SEQ ID NO:8, second row). The
first primer (SEQ ID NO:9, third row) anneals to the template (SEQ
ID NO:8, fourth row) and extends it by incorporating the
appropriate nucleotides. One of the nucleotides, in this case dGTP,
is labeled with a fluorescent dye. A sequence-specific
oligonucleotide probe hybridizes to the site of interest of SEQ ID
NO:9 to afford SEQ ID NO:10, fifth row. If the labeled guanine is
present (SEQ ID NO:10, last row), a FRET reaction occurs. The
energy emitted from the guanine is transferred to the label on the
probe. The energy emitted from the probe is detected by real-time
fluorescence detection.
Sequence CWU 1
1
10 1 96 DNA Artificial Sequence example for DNA sample 1 agtgtcttat
gttgtcagtg tagtctgtaa ggcaatgtct agtgattgat cgtgtagctg 60
taaagtgtag taagtagtgg gtatgtaaca tgttag 96 2 96 DNA Artificial
Sequence chemically treated DNA sample 2 agtgttttat gttgttagtg
tagtttgtaa ggtaatgttt agtgattgat cgtgtagttg 60 taaagtgtag
taagtagtgg gtatgtaata tgttag 96 3 56 DNA Artificial Sequence
labeled primer 3 tcacaaaata caacaatcac atcaaacatt ccattacaaa
tcactaacta gcacat 56 4 96 DNA Artificial Sequence labeled
oligonucleotide 4 tcacaaaata caacaatcac atcaaacatt ccattacaaa
tcactaacta gcacatcaac 60 atttcacatc attcatcacc catacattat acaatc 96
5 19 DNA Artificial Sequence primer oligonucleotide 5 gtgattgatc
gtgtagttg 19 6 22 DNA Artificial Sequence primer oligonucleotide 6
tagtgggtat gtaatatgtt ag 22 7 37 DNA Artificial Sequence example
for DNA sample 7 ttcaacatac agcatacgca ataagcaaat acctcac 37 8 37
DNA Artificial Sequence example for DNA sample 8 aagttgtatg
tcgtatgcgt tattcgttta tggagtg 37 9 30 DNA Artificial Sequence
labeled primer 9 ttcaacatac agcatacgca ataagcaaat 30 10 37 DNA
Artificial Sequence labeled DNA sample 10 ttcaacatac agcatacgca
ataagcaaat acctcac 37
* * * * *