U.S. patent application number 10/363345 was filed with the patent office on 2004-11-25 for method for determining the degree of methylation of defined cytosines in genomic dna in the sequence context 5'-cpg-3'.
Invention is credited to Berlin, Kurt, Gutig, David, Olek, Alexander, Piepenbrock, Christian.
Application Number | 20040234960 10/363345 |
Document ID | / |
Family ID | 26006939 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040234960 |
Kind Code |
A1 |
Olek, Alexander ; et
al. |
November 25, 2004 |
Method for determining the degree of methylation of defined
cytosines in genomic dna in the sequence context 5'-cpg-3'
Abstract
A method is described for the detection of the degree of
methylation of a specific cytosine in the sequence context
5'-CpG-3' of a genomic DNA sample. In the first step, the genomic
DNA is chemically treated in such a way that the cytosine bases are
converted to uracil, but not the 5-methylcytosine bases. Then
segments of the genomic DNA which contain the said specific
cytosine are amplified, whereby the amplified products are given a
detectable label and in the following steps the extent of
hybridization of the amplified products on two classes of
oligonucleotides is determined by detection of the label of the
amplified products, and a conclusion is made on the extent of
methylation of said specific cytosine in the genomic DNA sample
from the ratio of the labels detected on the two classes of
oligonucleotides as a consequence of the hybridization.
Inventors: |
Olek, Alexander; (Berlin,
DE) ; Piepenbrock, Christian; (Berlin, DE) ;
Berlin, Kurt; (Stahnsdorf, DE) ; Gutig, David;
(Berlin, DE) |
Correspondence
Address: |
KRIEGSMAN & KRIEGSMAN
665 FRANKLIN STREET
FRAMINGHAM
MA
01702
US
|
Family ID: |
26006939 |
Appl. No.: |
10/363345 |
Filed: |
March 3, 2003 |
PCT Filed: |
September 1, 2001 |
PCT NO: |
PCT/EP01/10074 |
Current U.S.
Class: |
435/6.11 ;
435/6.12; 435/91.2 |
Current CPC
Class: |
C12Q 2600/154 20130101;
C12Q 1/6883 20130101; C12Q 1/6827 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 1, 2000 |
DE |
100 43 826.1 |
Sep 5, 2000 |
DE |
100 44 543.8 |
Claims
1. A method for the detection of the degree of methylation of a
specific cytosine in the sequence context 5'-CpG-3' of a genomic
DNA sample, is hereby characterized in that a) the genomic DNA is
chemically treated, whereby the cytosine bases are converted to
uracil, but not the 5-methylcytosine bases; b) segments of the
genomic DNA, which contain said specific cytosine, are amplified,
whereby the amplified products are given a detectable label; c) the
amplified products are hybridized to two classes of
oligonucleotides and/or PNA oligomers, each of which class has at
least one member; d) the extent of hybridization of the amplified
products to the two classes of oligonucleotides and/or PNA
oligomers is determined by detection of the label of the amplified
products; e) a conclusion is made on the extent of methylation of
said specific cytosine in the genomic DNA sample from the ratio of
the labels detected for the two classes of oligonucleotides and/or
PNA oligomers as a consequence of the hybridization.
2. The method according to claim 1, further characterized in that a
hybridization of the amplified products is conducted in step c) on
two classes of oligomers (oligonucleotides and/or PNA oligomers),
each of which class has at least one member, whereby the oligomers
of the first class preferably hybridize to the sequence which
arises after the chemical treatment of the genomic DNA, if said
specific cytosine was present in the methylated state in the
genomic DNA and whereby the oligomers of the second class
preferably hybridize to the sequence which arises after the
chemical treatment of the genomic DNA, if said specific cytosine
was present in the unmethylated state in the genomic DNA.
3. The method according to claim 1, further characterized in that a
hybridization of the amplified products is conducted in step c) on
two classes of oligomers (oligonucleotides and/or PNA oligomers),
each of which class has at least one member, whereby the oligomers
of the first class preferably hybridize to the sequence which
arises after the chemical treatment of the genomic DNA, if said
specific cytosine was present in the methylated state in the
genomic DNA and less preferably hybridize to the sequence which
arises after the chemical treatment of the genomic DNA, if said
specific cytosine was present in the unmethylated state in the
genomic DNA, and whereby the oligomers of the second class
hybridize to the amplified product to be investigated essentially
independently of the degree of methylation of said specific
cytosine in the genomic DNA.
4. The method according to claim 1, further characterized in that a
hybridization of the amplified products is conducted in step c) on
two classes of oligomers (oligonucleotides and/or PNA oligomers),
each of which class has at least one member, whereby the oligomers
of the first class preferably hybridize to the sequence which
arises after the chemical treatment of the genomic DNA, if said
specific cytosine was present in the unmethylated state in the
genomic DNA and less preferably hybridize to the sequence which
arises after the chemical treatment of the genomic DNA, if said
specific cytosine was present in the methylated state in the
genomic DNA, and whereby the oligomers of the second class
hybridize to the amplified product to be investigated essentially
independently of the degree of methylation of said specific
cytosine in the genomic DNA.
5. The method according to claim 1, further characterized in that
the method is conducted not only with the genomic DNA sample, but
also logically with standard DNA in which it is known whether the
cytosine at said specific position is present in methylated or
unmethylated state, whereby the ratios of the labels detected on
the two classes of oligonucleotides, which are measured each time
with the unmethylated standard DNA according to claim 1, serve as a
calibration value for a degree of methylation of 0, and
correspondingly, the ratios of the labels detected on the two
classes of oligonucleotides, which are measured each time with the
methylated standard DNA according to claim 1, serve as a
calibration value for a degree of methylation of 1, and these
calibration values are used for the determination of the degree of
methylation of the genomic DNA sample.
6. The method according to claim 5, further characterized in that
additional known standard DNA samples, each of which has any known
degree of methylation of said specific cytosine, are used for
calibration.
7. The method according to claim 5, further characterized in that
the DNAs used as the standard are each labeled differently and the
amplified product from the genomic DNA is provided in turn with a
label that is different therefrom.
8. The method according to claim 1, further characterized in that
amplified products that are derived from different genomic DNA
samples are provided with different labels.
9. The method according to claim 1, further characterized in that
amplified products originating from the same genomic DNA samples
are provided with different labels in order to achieve an increase
of measurement accuracy by an averaging of the values obtained from
different detection methods.
10. The method according to claim 1, further characterized in that
said labels are fluorescent labels.
11. The method according to claim 1, further characterized in that
the chemical treatment is conducted with a solution of a bisulfite
(=hydrogen sulfite, disulfite).
12. The method according to claim 1, further characterized in that
oligonucleotides are used for the amplification, which comprise a
sequence segment of a chemically pretreated DNA which is at least
18 bases long of one of the sequences Seq. ID 1 to Seq. ID
40712.
13. The method according to claim 1, further characterized in that
in a hybridization step, oligonucleotides and/or peptide nucleic
acid (PNA) oligomers are used, which hybridize to a sequence
segment that is at least 9 bases long of a chemically pretreated
DNA according to one of the sequences Seq. ID 1 to Seq. ID 40712 or
correspond to this sequence, whereby the base sequence contains at
least one CpG dinucleotide and the CpG dinucleotide is found in
approximately the middle third of the oligomer.
14. The method according to claim 1, further characterized in that
said label is detected by its chemiluminescence, its UV absorption
or fluorescence polarization.
15. The method according to claim 1, further characterized in that
the labels are radionuclides.
16. The method according to claim 1, further characterized in that
the labels are removable mass labels, which are detected in a mass
spectrometer.
17. The method according to claim 1, further characterized in that
the PCR products as a whole or their characteristic fragments are
detected in the mass spectrometer and thus are clearly
characterized by their mass.
18. The method according to claim 1, further characterized in that
the oligomers (oligonucleotides and/or PNA oligomers) of one class
contain the sequence 5'-CG-3'.
19. The method according to claim 1, further characterized in that
the oligomers (oligonucleotides and/or PNA oligomers) of one class
contain the sequence 5'-TG-3' and/or the sequence 5'-CA-3'.
20. The method according to claims 18 or 19, further characterized
in that the oligomers (oligonucleotides and/or PNA oligomers) of
the first class contain the sequence 5'-CG-3' and the oligomers of
the second class contain the sequence 5'-TG-3' and/or the sequence
5'-CA-3'.
21. The method according to claim 1, further characterized in that
the oligomers of the first and the second classes are immobilized
on a common solid phase.
22. The method according to claim 11, further characterized in that
the oligonucleotides are arranged on a planar solid phase in a
rectangular or hexagonal grid and the site of specific
oligonucleotides on the solid phase is correlated with their
respective sequence.
23. The method according to claim 1, further characterized in that
the oligomers of the first and second classes are immobilized on
beads, which are coded with a set of separately detectable
labels.
24. The method according to claim 1, further characterized in that
step b) is conducted in two sub-steps as follows: a) a PCR
pre-amplification with at least one pair of primers of different
sequence which hybridize nonspecifically to a DNA sample pretreated
according to claim 1 and thus produce more than one amplified
product in the PCR step; b) a PCR amplification of the product
formed in the pre-amplification, with primers of different
sequence, which are each identical or inversely complementary to a
segment of the DNA sample ((+) strand or (-) strand) that has been
pretreated according to claim 1, and hybridize specifically to the
DNA to be amplified.
25. The method according to claim 1, further characterized in that
the amplification of several DNA segments is conducted in one
reaction vessel.
26. The method according to claim 1, further characterized in that
a heat-stable DNA polymerase is used for the amplification.
27. The method according to claim 1, further characterized in that
the primer oligonucleotides used for the amplification contain
either only the bases T, A and C or the bases T, A and G.
28. The method according to claim 1, further characterized in that
at least 10 CpG positions in different sequence context are
analyzed simultaneously.
29. The method according to claim 1, further characterized in that
at least 50 CpG positions in different sequence context are
analyzed simultaneously.
30. The method according to claim 1, further characterized in that
at least 100 CpG positions in different sequence context are
analyzed simultaneously.
31. The method according to claim 1, further characterized in that
at least 500 CpG positions in different sequence context are
analyzed simultaneously.
32. The method according to claim 1, further characterized in that
at least 1000 CpG positions in different sequence context are
analyzed simultaneously.
33. The method according to claim 1, whereby the genomic DNA sample
has been obtained from cell lines, blood, sputum, stool, urine,
cerebrospinal fluid, tissue embedded in paraffin, for example,
tissue from eyes, intestine, kidney, brain, heart, prostate, lung,
breast or liver, histological slides or all other possible
combinations thereof.
34. Use of a method according to claim 1 for the diagnosis and/or
prognosis of adverse events for patients or individuals, whereby
these adverse events belong to at least one of the following
categories: undesired drug interactions; cancer diseases; CNS
malfunctions, damage or disease; symptoms of aggression or
behavioral disturbances; clinical, psychological and social
consequences of brain damage; psychotic disturbances and
personality disorders; dementia and/or associated syndromes;
cardiovascular disease, malfunction and damage; malfunction, damage
or disease of the gastrointestinal tract; malfunction, damage or
disease of the respiratory system; lesion, inflammation, infection,
immunity and/or convalescence; malfunction, damage or disease of
the body as an abnormality in the development process; malfunction,
damage or disease of the skin, the muscles, the connective tissue
or the bones; endocrine and metabolic malfunction, damage or
disease; headaches or sexual malfunctions.
35. Use of a method according to claim 1 for the differentiation of
cell types or tissues of for investigation of cell
differentiation.
36. A kit, comprising a reagent containing bisulfite, primer
oligonucleotides for preparing the amplified products and/or
preferably oligonucleotides wherein the labels are fluorescent
labels, immobilized on a solid phase, as well as instructions for
conducting the method of claim 1.
37. Nucleic acids comprising a sequence segment at least 18 bases
long of a chemically pretreated DNA according to one of the
sequences Seq. ID 1 to Seq. ID 40712.
38. An oligomer (oligonucleotide or peptide nucleic acid (PNA)
oligomer) for the detection of the cytosine methylation state in
chemically pretreated DNA, each containing at least one base
sequence with a length of at least 9 nucleotides, which hybridizes
to a chemically pretreated DNA (Seq. ID 1 to Seq. ID 40712).
39. The oligomer according to claim 38, wherein the base sequence
contains at least one CpG dinucleotide.
40. The oligomer according to claim 39, further characterized in
that the cytosine of the CpG dinucleotide is found in approximately
the middle third of the oligomer.
41. A set of oligomers according to claim 39, comprising at least
one oligomer for at least one of the CpG dinucleotides of one of
the sequences of Seq. ID 1 to Seq. ID 40712.
42. The set of oligomers according to claim 41 containing at least
one oligomer for each of the CpG dinucleotides of one of the
sequences of Seq. ID 1 to Seq. ID 40712.
43. A set of at least two nucleic acids, which are utilized as
primer oligonucleotides for the amplification according to claim 1
of at least one of the sequences Seq. ID 1 to Seq. ID 40712 or
segments thereof.
44. The set of oligonucleotides according to claim 43, further
characterized in that at least one oligonucleotide is bound to a
solid phase.
45. A set of oligomer probes for the detection of the cytosine
methylation state and/or of single nucleotide polymorphisms (SNPs)
in chemically pretreated genomic DNA according to one of the
sequences Seq. ID 1 to Seq. ID 40712, containing at least ten of
the oligomers according to one of claims 38 to 40.
46. A method for the production of an arrangement of different
oligomers (an array) fixed on a support material for the analysis
of disorders related to the methylation state of the CpG
dinucleotides of one of the sequences Seq. ID 1 to Seq. ID 40712,
in which at least one oligomer according to one of claims 2 to 4 is
coupled to a solid phase.
47. An arrangement of different oligomers (an array) according to
one of claims 38 to 40, which is bound to a solid phase.
48. The array of different oligonucleotide and/or PNA oligomer
sequences according to claim 47, further characterized in that
these are arranged on a planar solid phase in the form of a
rectangular or hexagonal grid.
49. The array according to claim 47, further characterized in that
the solid phase surface is comprised of silicon, glass,
polystyrene, aluminum, steel, iron, copper, nickel, silver, or
gold.
50. A DNA and/or PNA array for the analysis of disorders related to
the methylation state of genes, which contains at least one nucleic
acid according to one of claims 38 to 40.
Description
[0001] The invention concerns a method for the detection of the
degree of methylation of a specific cytosine in the sequence
context 5'-CpG-3' of a genomic DNA sample.
[0002] The levels of observation that have been well studied in
molecular biology according to developments in methods in recent
years include the genes themselves, the transcription of these
genes into RNA and the translation to proteins therefrom. During
the course of development of an individual, which gene is turned on
and how the activation and inhibition of certain genes in certain
cells and tissues are controlled can be correlated with the extent
and nature of the methylation of the genes or of the genome. In
this regard, pathogenic states are also expressed by a modified
methylation pattern of individual genes or of the genome.
[0003] The present invention describes a method with which many
cytosine bases in a given DNA sample can be investigated
simultaneously for the presence of a methyl group at position 5 by
means of hybridization. It also describes nucleic acids,
oligonucleotides and PNA oligomers which are useful in order to
employ the method for the diagnosis of existing diseases or of
predisposition for [certain] diseases.
[0004] 5-Methylcytosine is the most frequent covalently modified
base in the DNA of eukaryotic cells. For example, it plays a role
in the regulation of transcription, in genetic imprinting and in
tumorigenesis. The identification of 5-methylcytosine as a
component of genetic information is thus of considerable interest.
5-Methylcytosine positions, however, cannot be identified by
sequencing, since 5-methylcytosine has the same base-pairing
behavior as cytosine. In addition, in the case of a PCR
amplification, the epigenetic information which is borne by the
5-methylcytosines is completely lost.
[0005] The methylation of CpG islands is often equated with
transcription inactivity. Although there is clear evidence that CpG
islands are to be found in promoters of genes, not all CpG islands
and methylation sites are localized in known promoters. In various
tissue-specific and imprinting genes, the CpG islands are localized
at considerable distance downstream of the start of transcription,
and also many genes possess multiple promoters. Methylation of CpG
dinucleotides has been detected as a causal factor for a number of
diseases. In contrast to classical mutations, DNA methylation
involves a mechanism, which describes a substitution on the base
without modifying the coding function of a gene. This interplay
between epigenetic modification and classical mutations plays an
important role in tumorigenesis. For example, focal
hypermethylation and generalized genomic demethylation are features
of many different tumor types. It is assumed that tumorigenesis and
tumor progression are caused, first of all, by hypermethylation of
induced mutation events, and secondly, by the turning off of genes
which control cellular proliferation, and/or the induced
reactivation of genes, which are [normally] used only for
embryological development, via demethylation.
[0006] In hereditable non-polyposis colorectal cancer, e.g., the
majority of mutation-negative cases of colon cancer are based
rather on the hypermethylation of the hMLH1 promoter and the
associated non-expression of hMLH1, a repair gene for erroneous
base pairings (Bevilacqua R A, Simpson A J, Methylation of the
hMLH1 promoter but no hMLH1 mutations in sporadic gastric
carcinomas with high-level microsatellite instability. Int J
Cancer. 2000 Jul. 15;87(2):200-3). In the pathogenesis of lung
cancer, the loss of expression is correlated with the methylation
of CpG islands in the promoter sequence of an RAS effector homolog.
(Dammann R, Li C, Yoon J H, Chin P L, Bates S, Pfeifer G P,
Nucleotide. Epigenetic inactivation of a RAS association domain
family protein from the lung tumour suppressor locus 3p21.3. Nat
Genet. July 2000;25(3):315-9). An epigenetic inactivation of the
LKB1 tumor supressor gene, including the hypermethylation of the
promoter, is associated with the Peutz-Jeghers syndrome (Esteller
M, Avizienyte E, Corn P G, Lothe R A, Baylin S B, Aaltonen L A,
Herman J G, Epigenetic inactivation of LKB1 in primary tumors
associated with the Peutz-Jeghers syndrome. Oncogene. 2000 Jan.
6;19(1):164-8).
[0007] A plurality of diseases, which are associated with
methylation, have in their etiology a close connection with the
tumor suppressor genes p16 or p15. Thus a relationship between
Mycosis fungoides and hypermethylation of the p16(INK4a) gene is
assumed (Navas I C, Ortiz-Romero P L, Villuendas R, Martinez P,
Garcia C, Gomez E, Rodriguez J L, Garcia D, Vanaclocha F, Iglesias
L, Piris M A, Algara P, p16(INK4a) gene alterations are frequent in
lesions of mycosis fungoides. Am J Pathol. May 2000;
156(5):1565-72). Also, there is a strong correlation between the
turning off of the transcription of the p16 gene in gastric
carcinoma and the de novo methylation of a few specific CpG sites
(Song S H, Jong H S, Choi H H, Kang S H, Ryu M H, Kim N K, Kim W H,
Bang Y J, Methylation of specific CpG sites in the promoter region
could significantly down-regulate p16(INK4a) expression in gastric
adenocarcinoma. Int J Cancer. 2000 Jul. 15;87(2):236-40). The
pathogenesis of cholangiocarcinoma, which is associated with
primary sclerosing cholangitis, has been related to the
inactivation of the p16 tumor suppressor gene, which is again
dependent on the methylation of the p16 promoter (Ahrendt S A,
Eisenberger C F, Yip L, Rashid A, Chow J T, Pitt H A, Sidransky D,
Chromosome 9p21 loss and p16 inactivation in primary sclerosing
cholangitis-associated cholangiocarcinoma. J Surg Res. 1999 Jun.
1;84(1):88-93). The inactivation of the p16 gene by
hypermethylation plays a role in the genesis of leukemia and in the
progression of acute lymphoblastic leukemia (Nakamura M, Sugita K,
Inukai T, Goi K, Iijima K, Tezuka T, Kojika S, Shiraishi K,
Miyamoto N, Karakida N, Kagami K, O-Koyama T, Mori T, Nakazawa S,
p16/MTS1/INK4A gene is frequently inactivated by hypermethylation
in childhood acute lymphoblastic leukemia with 11 q23
translocation. Leukemia. June 1999;13(6):884-90). In addition, it
is postulated that the hypermethylation of the p16 and p15 genes
plays a decisive role in the tumorigenesis of multiple myeloma (Ng
M H, Wong I H, Lo K W, DNA methylation changes and multiple
myeloma. Leuk Lymphoma. August 1999;34(5-6):463-72). The VHL gene,
which is inactivated by methylation, appears to participate in
predisposition to renal carcinoma (Glavac D, Ravnik-Glavac M, Ovcak
Z, Masera A, Genetic changes in the origin and development of renal
cell carcinoma (RCC). Pflugers Arch. 1996;431(6 Suppl 2):R193-4). A
divergent methylation of the 5'-CpG island may participate in
nasopharyngeal carcinoma, possibly by the inactivation of
transcription of the p16 gene (Lo K W, Cheung S T, Leung S F, van
Hasselt A, Tsang Y S, Mak K F, Chung Y F, Woo J K, Lee J C, Huang D
P, Hypermethylation of the p16 gene in nasopharyngeal carcinoma.
Cancer Res. 1996 Jun. 15;56(12):2721-5). An inactivation of the p16
protein was detected in liver cell carcinoma. Promoter
hypermethylation and homozygous deletions are the most frequent
mechanisms here (Jin M, Piao Z, Kim N G, Park C, Shin E C, Park J
H, Jung H J, Kim C G, Kim H, p16 is a major inactivation target in
hepatocellular carcinoma. Cancer. 2000 Jul. 1;89(1):60-8). DNA
methylation as a control of gene expression was detected for the
BRCA1 gene for breast cancer (Magdinier F, Billard L M, Wittmann G,
Frappart L, Benchaib M, Lenoir G M, Guerin J F, Dante R, Regional
methylation of the 5' end CpG island of BRCA1 is associated with
reduced gene expression in human somatic cells FASEB J. August
2000; 14(11):1585-94). A correlation between methylation and
non-Hodgkin's lymphoma is also assumed (Martinez-Delgado B, Richart
A, Garcia M J, Robledo M, Osorio A, Cebrian A, Rivas C, Benitez J,
Hypermethylation of P16ink4a and P15ink4b genes as a marker of
disease in the follow-up of non-Hodgkin's lymphomas. Br J Haematol.
April 2000;109(1):97-103). CpG methylation also brings about the
progression of T-cell leukemia, which is related to a decreased
expression of the CDKN2A gene (Nosaka K, Maeda M, Tamiya S, Sakai
T, Mitsuya H, Matsuoka M, Increasing methylation of the CDKN2A gene
is associated with the progression of adult T-cell leukemia. Cancer
Res. 2000 Feb. 15;60(4):1043-8). An increased methylation of the
CpG islands was established in bladder cancer (Salem C, Liang G,
Tsai Y C, Coulter J, Knowles M A, Feng A C, Groshen S, Nichols P W,
Jones P A, Progressive increases in de novo methylation of CpG
islands in bladder cancer. Cancer Res. 2000 May 1;60(9):2473-6).
Transcription inactivation by esophageal squamous cell carcinomas
has been related to the methylation of the FHIT gene, which is
associated with the progression of the disease (Shimada Y, Sato F,
Watanabe G, Yamasaki S, Kato M, Maeda M, Imamura M, Loss of fragile
histidine triad gene expression is associated with progression of
esophageal squamous cell carcinoma, but not with the patient's
prognosis and smoking history. Cancer. 2000 Jul. 1;89(1):5-11).
Neutral endopeptidase 24.11 (NEP) inactivates the increase of
neuropeptides which participate in the growth of
androgen-independent prostate cancer. A loss of NEP expression by
hypermethylation of the NEP promotors may contribute to the
development of neuropeptide-stimulated, androgen-independent
prostate cancer (Usmani B A, Shen R, Janeczko M, Papandreou C N,
Lee W H, Nelson W G, Nelson J B, Nanus D M, Methylation of the
neutral endopeptidase gene promoter in human prostate cancers. Clin
Cancer Res. May 2000;6(5):1664-70). Adrenocortical tumors in adults
display structural abnormalities in the tumor DNA. Among other
things, these abnormalities contain an overexpression of the IGF2
gene in correlation with a demethylation of the DNA at this locus
(Wilkin F, Gagne N, Paquette J, Oligny L L, Deal C, Pediatric
adrenocortical tumors: molecular events leading to insulin-like
growth factor II gene overexpression. J Clin Endocrinol Metab. May
2000; 85(5):2048-56. Review). It is assumed that DNA methylations
in several exons in the retinoblastoma gene contribute to the
disease (Mancini D, Singh S, Ainsworth P, Rodenhiser D,
Constitutively methylated CpG dinucleotides as mutation hot spots
in the retinoblastoma gene (RB1). Am J Hum Genet. July 1997;
61(1):80-7). In chronic myeloid leukemia, a relationship is
suspected between the deregulation of the p53 gene and a change in
the methylation pattern with progression of the disease (Guinn B A,
Mills K I, p53 mutations, methylation and genomic instability in
the progression of chronic myeloid leukaemia. Leuk Lymphoma. July
1997;26(34):211-26). A connection with methylation has also been
detected for acute myeloid leukemia (Melki J R, Vincent P C, Clark
S J. Concurrent DNA hypermethylation of multiple genes in acute
myeloid leukemia. Cancer Res. 1999 Aug. 1;59(15):3730-40). A
tumor-specific methylation site in the Wilms tumor suppressor gene
has been identified (Kleymenova E V, Yuan X, LaBate M E, Walker C
L, Identification of a tumor-specific methylation site in the Wilms
tumor suppressor gene. Oncogene. 1998 Feb. 12;16(6):713-20). In
Burkitt's lymphoma, several promotors have a complete CpG
methylation (Tao Q, Robertson K D, Manns A, Hildesheim A, Ambinder
R F, Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma:
molecular analysis of primary tumor tissue. Blood. 1998 Feb.
15;91(4):1373-81). It is assumed that DNA methylation plays a role
in thyroid carcinoma (Venkataraman G M, Yatin M, Marcinek R, Ain K
B, Restoration of iodide uptake in dedifferentiated thyroid
carcinoma: relationship to human Na+/I-symporter gene methylation
status. J Clin Endocrinol Metab. July 1999; 84(7):2449-57).
[0008] Not only are many cancer diseases associated with
methylation, but there are also many other diseases that are
related to methylation. Investigations of inflammatory arthritis
have indicated that this disease is associated with a
hypomethylation of genomic DNA (Kim Y I, Logan J W, Mason J B,
Roubenoff R, DNA hypomethylation in inflammatory arthritis:
reversal with methotrexate. J Lab Clin Med. August
1996;128(2):165-72). A methylation-regulated expression has been
detected for the ICF syndrome (Kondo T, Bobek M P, Kuick R, Lamb B,
Zhu X, Narayan A, Bourc'his D, Viegas-Pequignot E, Ehrlich M,
Hanash S M, Whole-genome methylation scan in ICF syndrome:
hypomethylation of nonsatellite DNA repeats D4Z4 and NBL2). The
participation of methylation is suspected in systemic lupus
erythematosus (Vallin H, Perers A, Alm G V, Ronnblom L,
Anti-double-stranded DNA antibodies and immunostimulatory plasmid
DNA in combination mimic the endogenous IFN-alpha inducer in
systemic lupus erythematosus. J Immunol. December
1999;163(11):6306-13); and there may also be a relationship between
the Duchenne muscular dystrophy gene and a CpG-rich island
(Banerjee S, Singh P B, Rasberry C, Cattanach B M, Embryonic
inheritance of the chromatin organisation of the imprinted H19
domain in mouse spermatozoa. Mech Dev. February 2000;90(2):217-26;
Burmeister M, Lehrach H, Long-range restriction map around the
Duchenne muscular dystrophy gene. Nature. 1986 Dec.
11-17;324(6097):582-5). An epigenetic effect, which involves the
hypomethylation of the amyloid precursor protein [gene], which is
related to the development of the disease, is suspected in
Alzheimer's disease (West R L, Lee J M, Maroun L E, Hypomethylation
of the amyloid precursor protein gene in the brain of an
Alzheimer's disease patient. J Mol Neurosci. 1995;6(2):141-6). The
methylation state also plays an important role at the chromosomal
level. For example, in mental retardation syndromes, which are
coupled with the fragility of the X chromosome, the degree of
chromosomal fragility is determined by the methylation (de Muniain
A L, Cobo A M, Poza J J, Saenz A, [Diseases due to instability of
DNA]. Neurologia. December 1995;10 Suppl 1:12-9).
[0009] A relatively new method that in the meantime has become the
most widely used method for investigating DNA for 5-methylcytosine
is based on the specific reaction of bisulfite with cytosine,
which, after subsequent alkaline hydrolysis, is then converted to
uracil, which corresponds in its base-pairing behavior to
thymidine. In contrast, 5-methylcytosine is not modified under
these conditions. Thus, the original DNA is converted so that
methylcytosine, which originally cannot be distinguished from
cytosine by its hybridization behavior, can now be detected by
"standard" molecular biology techniques as the only remaining
cytosine, for example, by amplification and hybridization or
sequencing. All of these techniques are based on base pairing,
which is now fully utilized. The prior art, which concerns
sensitivity, is defined by a method that incorporates the DNA to be
investigated in an agarose matrix, so that the diffusion and
renaturation of the DNA is prevented (bisulfite reacts only on
single-stranded DNA) and all precipitation and purification steps
are replaced by rapid dialysis. (Olek, A. et al., Nucl. Acids Res.
1996, 24, 5064-5066). Individual cells can be investigated by this
method, which illustrates the potential of the method. Of course,
up until now, only individual regions of up to approximately 3000
base pairs long have been investigated; a global investigation of
cells for thousands of possible methylation analyses is not
possible. Of course, this method also cannot reliably analyze very
small fragments of small quantities of sample. These are lost
despite the protection from diffusion through the matrix.
[0010] An overview of other known possibilities for detecting
5-methylcytosines can be derived from the following review article:
Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998,
26, 2255.
[0011] The bisulfite technique has been previously applied only in
research, with a few exceptions (e.g., Zechnigk, M. et al., Eur. J.
Hum. Gen. 1997, 5, 94-98). However, short, specific segments of a
known gene have always been amplified after a bisulfite treatment,
and either completely sequenced (Olek, A. and Walter, J., Nat.
Genet. 1997, 17, 275-276) or individual cytosine positions have
been detected by a primer extension reaction (Gonzalgo, M. L. and
Jones, P. A., Nucl. Acids Res. 1997, 25, 2529-2531, WO-Patent
95-00669) or an enzyme step (Xiong, Z. and Laird, P. W., Nucl.
Acids. Res. 1997, 25, 2532-2534). Detection by hybridization has
also been described (Olek et al., WO-A 99/28,498).
[0012] Other publications which are concerned with the application
of the bisulfite technique for the detection of methylation in the
case of individual genes are: Xiong, Z. and Laird, P. W. (1997),
Nucl. Acids Res. 25, 2532; Gonzalgo, M. L. and Jones, P. A. (1997),
Nucl. Acids Res. 25, 2529; Grigg, S. and Clark, S. (1994),
Bioassays 16, 431; Zeschnik, M. et al. (1997), Human Molecular
Genetics 6, 387; Teil, R. et al. (1994), Nucl. Acids Res. 22, 695;
Martin, V. et al. (1995), Gene 157, 261; WO-A 97/46,705, WO-A
95/15,373 and WO-A 95/45,560.
[0013] A review of the prior art in oligomer array production can
be taken from the special edition of Nature Genetics that appeared
in January 1999 (Nature Genetics Supplement, Volume 21, January
1999) and the literature cited therein.
[0014] Probes with multiple fluorescent labels have been used for
scanning an immobilized DNA array. Particularly suitable for
fluorescent labels is the simple introduction of Cy3 and Cy5 dyes
at the 5'-OH of the respective probe. The fluorescence of the
hybridized probes is detected, for example, by means of a confocal
microscope. The dyes Cy3 and Cy5, among many others, are
commercially available.
[0015] Matrix-assisted laser desorptions/ionization mass
spectrometry (MALDI-TOF) is a very powerful development for the
analysis of biomolecules (Karas, M. and Hillenkamp, F. (1988),
Laser desorption ionization of proteins with molecular masses
exceeding 10,000 daltons. Anal. Chem. 60: 2299-2301). An analyte is
embedded in a light-absorbing matrix. The matrix is vaporized by a
short laser pulse and the analyte molecule is transported
unfragmented into the gaseous phase. The analyte is ionized by
collisions with matrix molecules. An applied voltage accelerates
the ions in a field-free flight tube. Ions are accelerated to
varying degrees based on their different masses. Smaller ions reach
the detector sooner than large ions.
[0016] MALDI-TOF spectrometry is excellently suitable for the
analysis of peptides and proteins. The analysis of nucleic acids is
somewhat more difficult (Gut, I. G. and Beck, S. (1995)), DNA and
Matrix Assisted Laser Desorption Ionization Mass Spectrometry.
Molecular Biology: Current Innovations and Future Trends 1:
147-157). For nucleic acids, the sensitivity is approximately 100
times poorer than for peptides and decreases overproportionally
with increasing fragment size. For nucleic acids, which have a
backbone with a multiple negative charge, the ionization process
via the matrix is basically less efficient. In MALDI-TOF
spectrometry, the choice of matrix plays an imminently important
role. Several very powerful matrices, which produce a very fine
crystallization, have been found for the desorption of peptides. In
the meantime, several effective matrices have been developed for
DNA, but the difference in sensitivity has not been reduced
thereby. The difference in sensitivity can be reduced by modifying
the DNA chemically in such a way that it resembles a peptide.
Phosphorothioate nucleic acids, in which the usual phosphates of
the backbone are substituted by thiophosphates, can be converted by
simple alkylation chemistry to a charge-neutral DNA (Gut, I. G. und
Beck, S. (1995), A procedure for selective DNA alkylation and
detection by mass spectrometry. Nucleic Acids Res. 23: 1367-1373).
The coupling of a charge tag to this modified DNA results in an
increase in sensitivity of the same order of magnitude as is found
for peptides. Another advantage of charge tagging is the increased
stability of the analysis in the presence of impurities, which make
the detection of unmodified substrates very difficult.
[0017] Genomic DNA is obtained from DNA of cells, tissue or other
test samples by standard methods. This standard methodology is
found in references such as Fritsch and Maniatis, eds., Molecular
Cloning: A Laboratory Manual, 1989.
[0018] The present invention will present a particularly efficient
and reliable method, which permits investigating many cytosine
bases in a given DNA sample simultaneously for the presence of a
methyl group at position 5 by means of hybridization.
Oligonucleotides and PNA oligomers are also presented for this
purpose, which are particularly suitable for using the method for
the diagnosis of existing diseases and of predisposition for
diseases by analysis of a set of genetic and/or epigenetic
parameters.
[0019] Genetic parameters in the sense of this invention are
mutations and polymorphisms of the claimed nucleic acids (Seq. ID 1
to Seq. ID 40712) and additional sequences necessary for their
regulation. Particularly designated as mutations are insertions,
deletions, point mutations, inversions and polymorphisms and
particularly preferred are SNPs (single nucleotide polymorphisms).
Polymorphisms, however, can also be insertions, deletions or
inversions.
[0020] Epigenetic parameters in the sense of this invention are
particularly cytosine methylations and other chemical modifications
of DNA bases of the claimed nucleic acids (Seq. ID 1 to Seq. ID
40712) and additional sequences necessary for their regulation.
Other epigenetic parameters, for example, are the acetylation of
histones, although this cannot be directly analyzed with the
described method; however, it is correlated in turn with DNA
methylation.
[0021] The present method serves for the detection of the degree of
methylation of at least one specific cytosine in the sequence
context 5'-CpG-3' of a genomic DNA sample. The method is
particularly preferably used for the simultaneous detection of many
different methylation positions.
[0022] The object is solved according to the invention by a method
for the detection of the degree of methylation of a specific
cytosine in the sequence context 5'-CpG-3' of a genomic DNA sample,
which is characterized in that
[0023] a) the genomic DNA is treated, whereby the cytosine bases
are converted to uracil, but not the 5-methylcytosine bases;
[0024] b) segments of the genomic DNA, which contain said specific
cytosine, are amplified, whereby the amplified products are given a
detectable label;
[0025] c) the amplified products are hybridized to two classes of
oligonucleotides and/or PNA oligomers, each of which [class] has at
least one member;
[0026] d) the extent of hybridization of the amplified products on
the two classes of oligonucleotides is determined by detection of
the label of the amplified products;
[0027] e) a conclusion is made on the extent of methylation of said
specific cytosine in the genomic DNA sample from the ratio of the
labels detected on the two classes of oligonucleotides as a
consequence of the hybridization.
[0028] A method is particularly preferred in which a hybridization
of the amplified products is conducted in step c) on two classes of
oligomers (oligonucleotides and/or PNA oligomers), each of which
[class] has at least one member, whereby the oligomers of the first
class preferably hybridize to the sequence which arises from the
chemical treatment of the genomic DNA, if said specific cytosine
was present in the methylated state in the genomic DNA, and whereby
the oligomers of the second class preferably hybridize to the
sequence which arises from the chemical treatment of the genomic
DNA if said specific cytosine was present in the unmethylated state
in the genomic DNA. One of these two classes of oligomers, for
example, can be formed by oligonucleotides which contain a CG in
the middle, and the other class can be formed by oligonucleotides
which have a TG (or a CA, in the counterstrand) in the middle. The
remaining segments of the oligomer sequences should preferably be
the same in the two classes. In this case, oligonucleotides of the
first class hybridize to the sequence (around the specific cytosine
to be investigated) if it was present in the methylated state
before bisulfite conversion, and vice versa, those of the second
class would hybridize to the sequence if it was present in the
unmethylated state before the bisulfite conversion.
[0029] A method is also particularly preferred in which a
hybridization of the amplified products is conducted in step c) on
two classes of oligomers (oligonucleotides and/or PNA oligomers),
each of which [class] has at least one member, whereby the
oligomers of the first class preferably hybridize to the sequence
which arises after the chemical treatment of the genomic DNA if
said specific cytosine was present in the methylated state in the
genomic DNA and less preferably hybridize to the sequence which
arises after the chemical treatment of the genomic DNA if said
specific cytosine was present in the unmethylated state in the
genomic DNA, and whereby the oligomers of the second class
hybridize to the amplified product to be investigated essentially
independently of the degree of methylation of said specific
cytosine in the genomic DNA.
[0030] Correspondingly, a method is also particularly preferred in
which a hybridization of the amplified products is conducted in
step c) on two classes of oligomers (oligonucleotides and/or PNA
oligomers), each of which [class] has at least one member, whereby
the oligomers of the first class preferably hybridize to the
sequence which arises after the chemical treatment of the genomic
DNA if said specific cytosine was present in the unmethylated state
in the genomic DNA and less preferably hybridize to the sequence
which arises after the chemical treatment of the genomic DNA if
said specific cytosine was present in the methylated state in the
genomic DNA, and whereby the oligomers of the second class
hybridize to the amplified product to be investigated essentially
independently of the degree of methylation of said specific
cytosine in the genomic DNA.
[0031] Thus, in these cases, the second class of oligomers
hybridizes to the amplified product without producing an essential
methylation specificity, and thus accordingly, preferably to a
position of the amplified product which does not correspond to
methylatable cytosine positions. Thus, only the concentration of
the amplified product is determined by the intensity of
hybridization. In this regard, hybridization to the first class of
oligonucleotides results as a function of the degree of methylation
of the specific cytosine to be investigated.
[0032] It is preferred that the method is conducted not only with
the genomic DNA sample, but also logically with standard DNA in
which it is known whether the cytosine at said specific position is
present in methylated or unmethylated state, whereby the ratios of
the labels detected on the two classes of oligonucleotides, which
are measured each time with the unmethylated standard DNA, serve as
a calibration value for a degree of methylation of 0, and
correspondingly the ratios of the labels detected on the two
classes of oligonucleotides, which are measured each time with the
methylated standard DNA, serve as a calibration value for a degree
of methylation of 1, and these calibration values are used for the
determination of the degree of methylation of the genomic DNA
samples.
[0033] It is particularly preferred that additional known standard
DNA samples, each of which has any known degree of methylation of
said specific cytosine, are used for calibration.
[0034] The standard DNA samples used and the samples (the amplied
product prepared from a genomic DNA) are each preferably given a
different label. The standard DNA samples used are each preferably
labeled in turn with different labels.
[0035] A method is also particularly preferred in which amplified
products originating from different genomic DNA samples are
provided with different labels. In this case, it is possible to
measure different samples simultaneously with one set of
oligonucleotides of the two classes, for example, on an oligomer
array which contains oligonucleotides of the two classes.
[0036] A method is also particularly preferred, in which amplified
products originating from the same genomic DNA samples are provided
with different labels in order to achieve an increase of
measurement accuracy by an averaging of the values obtained from
different detection methods. For example, this can be carried out
by labeling with different fluorescent dyes. In this case, the
measurement is conducted with a fluorescence scanner, which
provides several channels for the measurement of individual
emission wavelengths of the fluorescent dyes.
[0037] Accordingly, a method is also particularly preferred, in
which the labels are fluorescent labels.
[0038] According to the invention, it is further preferred that
said label is a fluorescent label. It is preferred that said label
is detected by chemiluminescence, its UV absorption or fluorescence
polarization.
[0039] It is particularly preferred according to the invention that
the DNA treatment in step a) is conducted with a solution of a
bisulfite (=hydrogen sulfite, disulfite). A method is also
particularly preferred, in which oligonucleotides are used for the
amplification, which comprise a sequence segment of a chemically
pretreated DNA which is at least 18 bases long according to one of
the [sequences] Seq. ID 1 to Seq. ID 40712. It is assured that
primers complementary to the bisulfite-treated DNA are used, which
can amplify regulatory regions (CpG islands) which can then be
investigated with respect to methylation.
[0040] A method is also particularly preferred, in which, in a
hybridization step, oligonucleotides and/or peptide nucleic acid
(PNA) oligomers are used, which hybridize to a sequence segment
that is at least 9 bases long of a chemically pretreated DNA
according to one of the [sequences] Seq. ID 1 to Seq. ID 40712 or
correspond to this segment, whereby the base sequence contains at
least one CpG dinucleotide and the CpG dinucleotide is found in
approximately the middle third of the oligomer. These
oligonucleotides are suitable for investigating specific CpG
positions with respect to their degree of methylation according to
the method of the invention. They preferably bind to the amplified
products of treated DNA, which originates from a genomic DNA sample
methylated at the respective cytosine positions.
[0041] It is also preferred that the labels are radionuclides.
[0042] It is further preferred that the labels are removable mass
labels, which are detected in a mass spectrometer. It is
particularly preferred according to the invention that the PCR
products as a whole or their characteristic fragments are detected
in the mass spectrometer and thus are clearly characterized by
their mass.
[0043] It is particularly preferred according to the invention that
the oligomers (oligonucleotides and/or PNA oligomers) of one class
contain the sequence 5'-CG-3'.
[0044] It is particularly preferred according to the invention that
the oligomers (oligonucleotides and/or PNA oligomers) of one class
contain the sequence 5'-TG-3' and/or the sequence 5'-CA-3'.
[0045] It is also particularly preferred that the oligonucleotides
of the first class contain the sequence 5'-CG-3' and the
oligonucleotides of the second class contain the sequence 5'-TG-3'
and/or the sequence 5'-CA-3'.
[0046] It is also preferred that the oligonucleotides of the first
and of the second classes are immobilized on a common solid phase.
It is also preferred that the oligonucleotides are arranged on a
planar solid phase in a rectangular or hexagonal grid and the site
of specific oligonucleotides on the solid phase is correlated with
their respective sequence.
[0047] It is also particularly preferred that the oligomers of the
first and second classes are immobilized on beads, which are coded
with a set of separately detectable labels. The latter serve for
identifying the bead, i.e., the sequence bound to the bead in
question. The amplified products bound to the beads are then
identified by means of other labels, which are bound to the
amplified products. Instruments for conducting such measurements
based on beads are offered, for example, by the Luminex
company.
[0048] A method is most particularly preferred according to the
invention, wherein step b) is conducted in two sub-steps as
follows:
[0049] a) a PCR pre-amplification with at least one pair of primers
of different sequence which hybridize nonspecifically to a DNA
sample pretreated according to claim 1 and thus produce more than
one amplified product in the PCR step;
[0050] b) a PCR amplification of the product formed in the
pre-amplification, with primers of different sequence, which are
each identical or inversely complementary to a segment of the DNA
sample [(+) strand or (-) strand] that has undergone pretreatment
according to claim 1, and hybridize specifically to the DNA to be
amplified.
[0051] In connection with this invention, hybridization is
understood as a hybridization of two single DNA strands that are
completely inversely complementary to each other according to
Watson-Crick rules without the occurrence of an erroneous base
pairing. Uracil is considered in this respect as thymine.
[0052] It is also preferred according to the invention that the
amplification of several DNA segments is conducted in one reaction
vessel.
[0053] It is further preferred according to the invention that a
heat-stable DNA polymerase is used for the amplification. It is
also particularly preferred that the primer oligonucleotides used
for the amplification contain either only the bases T, A and C or
the bases T, A and G.
[0054] It is also preferred that at least 10 CpG positions in
different sequence context are analyzed simultaneously. It is
particularly preferred that at least 50 CpG positions in different
sequence context are analyzed simultaneously. It is even more
particularly preferred that at least 100 CpG positions in different
sequence context are analyzed simultaneously. It is even more
preferred that at least 500 CpG positions in different sequence
context are analyzed simultaneously. It is most preferable that at
least 1000 CpG positions in different sequence context are analyzed
simultaneously.
[0055] The method is preferred according to the invention, whereby
the genomic DNA sample has been obtained from cell lines, blood,
sputum, stool, urine, cerebrospinal fluid, tissue embedded in
paraffin, for example, tissue from eyes, intestine, kidney, brain,
heart, prostate, lung, breast or liver, histological slides or all
other possible combinations thereof.
[0056] The use of a method according to the invention is preferred
for the diagnosis and/or prognosis of adverse events for patients
or individuals, whereby these adverse events belong to at least one
of the following categories: undesired drug interactions; cancer
diseases; CNS malfunctions, damage or disease; symptoms of
aggression or behavioral disturbances; clinical, psychological and
social consequences of brain damage; psychotic disturbances and
personality disorders; dementia and/or associated syndromes;
cardiovascular disease, malfunction and damage; malfunction, damage
or disease of the gastrointestinal tract; malfunction, damage or
disease of the respiratory system; lesion, inflammation, infection,
immunity and/or convalescence; malfunction, damage or disease of
the body as an abnormality in the development process; malfunction,
damage or disorder of the skin, the muscles, the connective tissue
or the bones; endocrine and metabolic malfunction, damage or
disease; headaches or sexual malfunctions.
[0057] The use of a method according to the invention is also
preferred for distinguishing cell types or tissues or for
investigating cell differentiation.
[0058] The subject of the present invention is [also] a kit
comprising a reagent containing bisulfite, primer oligonucleotides
for the production of the amplified products and/or preferably
oligonucleotides immobilized to a solid phase as well as
instructions for conducting the method according to the invention.
The primer oligonucleotides and the immobilized oligonucleotides,
as described above, are derived from the [sequences] Seq. IDs 1 to
40712.
[0059] This genomic DNA sample has been obtained preferably from
cell lines, blood, sputum, stool, urine, cerebrospinal fluid,
tissue embedded in paraffin, for example, tissue from eyes,
intestine, kidney, brain, heart, prostate, lung, breast or liver,
histological slides or all other possible combinations thereof.
[0060] In this method in the first step, a genomic DNA sample is
treated in such a way that except for the 5-methylcytosine bases,
all cytosine bases are converted to uracil. This chemical treatment
is preferably conducted with the solution of a bisulfite (=hydrogen
sulfite, disulfite). This step of the method can be conducted not
only with the genomic DNA sample, but also preferably and logically
with standard DNA in which it is known whether the cytosine at said
specific position is present in methylated or unmethylated
state.
[0061] In the second step of the method, the segments of the
genomic DNA that contain said specific cytosine are amplified. This
step can be particularly preferably conducted in two sub-steps:
[0062] 1. First, a PCR pre-amplification is conducted with at least
one pair of primers of different sequence, which hybridize to a
chemically pretreated DNA. This treatment was chemically conducted
in such a way that the cytosine bases were converted to uracil, but
not the 5-methylcytosine bases.
[0063] 2. A PCR amplification of the product formed in the
pre-amplification is conducted with primers of different sequence.
These primers are identical or inversely complementary to a segment
of the chemically pretreated DNA [(+) strand or (-) strand] and
specifically hybridize to the DNA to be amplified. The amplified
products preferably contain a detectable label.
[0064] In the following third step of the method, a hybridization
of the amplified products takes place on preferably two classes of
oligonucleotides, each of which [class] has at least one member. In
a particularly preferred variant of the method, the
oligonucleotides of the first class contain the sequence 5'-CG-3'
and the oligonucleotides of the second class contain the sequence
5'-TG-3' and/or the sequence 5'-CA-3'. The oligonucleotides of the
first and the second classes are preferably immobilized on a common
solid phase. The oligonucleotides are arranged on a planar solid
phase in a rectangular or hexagonal grid and the site of specific
oligonucleotides on the solid phase is correlated with their
respective sequence.
[0065] The oligonucleotides of the first class preferably hybridize
to the sequence which arises from the chemical treatment of the
genomic DNA if said specific cytosine was present in the methylated
state in the genomic DNA. The oligonucleotides of the second class
preferably hybridize to the sequence which arises from the chemical
treatment of the genomic DNA if said specific cytosine was present
in the unmethylated state in the genomic DNA.
[0066] The amplification of several DNA segments is particularly
preferably conducted in one reaction vessel. The amplification is
preferably conducted with the polymerase chain reaction (PCR),
wherein a heat-stable DNA polymerase is preferably used.
[0067] The primer oligonucleotides used for the amplification
contain preferably either only the bases T, A and C or the bases T,
A and G.
[0068] In the fourth step of the method, the extent of
hybridization of the amplified products on the two classes of
oligonucleotides is determined by detection of the labels of the
amplified products. The labels are particularly preferably
fluorescent labels, radionuclides, or removable mass labels, which
are detected in a mass spectrometer. The labels are preferably also
detected by chemiluminescence, UV absorption or fluorescence
polarization. The PCR products can also preferably be detected as a
whole or as their characteristic fragments in the mass
spectrometer. Thus the PCR products are clearly characterized by
their mass.
[0069] In the last step of the method, a conclusion is made on the
extent of methylation of said specific cytosine in the genomic DNA
sample from the ratio of the labels detected on the two classes of
oligonucleotides as a consequence of the hybridization.
[0070] The ratios of the labels detected on the two classes of
oligonucleotides, which are measured each time with the
unmethylated standard DNA, preferably serve as a calibration value
for a degree of methylation of 0.
[0071] Correspondingly, the ratios of the labels detected on the
two classes of oligonucleotides, which are measured each time with
the methylated standard DNA, preferably serve as a calibration
value for a degree of methylation of 1. The calibration values are
particularly preferably used for the determination of the degree of
methylation of the genomic DNA samples.
[0072] Preferably, additional known standard DNA samples, each of
which has any known degree of methylation of said specific
cytosine, are also used for the calibration.
[0073] The method is further characterized in that preferably at
least 10 CpG positions in different sequence context are analyzed
simultaneously. In addition, preferably at least 50 CpG positions
in different sequence context can be analyzed simultaneously. It is
also preferred that at least 100 CpG positions in different
sequence context are analyzed simultaneously. The simultaneous
analysis of at least 500 CpG positions in different sequence
context is very much preferred. The simultaneous analysis of at
least 1000 CpG positions in different sequence context is finally
particularly preferred.
[0074] The described method is preferably used for the diagnosis
and/or prognosis of adverse events for patients or individuals,
whereby these adverse events belong to at least one of the
following categories: undesired drug interactions; cancer diseases;
CNS malfunctions, damage or disease; symptoms of aggression or
behavioral disturbances; clinical, psychological and social
consequences of brain damage; psychotic disturbances and
personality disorders; dementia and/or associated syndromes;
cardiovascular disease, malfunction and damage; malfunction, damage
or disease of the gastrointestinal tract; malfunction, damage or
disease of the respiratory system; lesion, inflammation, infection,
immunity and/or convalescence; malfunction, damage or disease of
the body as an abnormality in the development process; malfunction,
damage or disorder of the skin, the muscles, the connective tissue
or the bones; endocrine and metabolic malfunction, damage or
disease; headaches or sexual malfunctions.
[0075] The present method is particularly preferably used for
distinguishing cell types or tissues or for investigating cell
differentiation.
[0076] By determining the hybridization ratios between the two
classes of oligonucleotides utilized (e.g., containing CG/TG), the
method is not dependent on the intensity of the total hybridization
of unknown tissue samples.
[0077] Unmethylated and methylated reference samples are utilized
as standards for calibrating unknown tissue samples.
[0078] A component of this method is also a kit, which comprises a
reagent containing bisulfite, primer oligonucleotides for the
production of amplified products and/or preferably oligonucleotides
immobilized on a solid phase. The oligonucleotides (first class)
comprise the sequence 5'-CG-3'. The oligonucleotides (second class)
comprise the sequence 5'-TG-3' and/or the sequence 5'-CA-3'.
Instructions for conducting the method are also included in the
kit.
[0079] The subject of the present invention is also nucleic acids
that are particularly suitable for conducting the method.
[0080] The subject of the invention is also a set of at least 10
oligomer probes (oligonucleotides and/or PNA oligomers), which
serve for the detection of the cytosine methylation state in
chemically pretreated genomic DNA (Seq. ID 1 to Seq. ID 40712). The
analysis of a set of genetic and/or epigenetic parameters for the
diagnosis of existing diseases or for the diagnosis of
predisposition to specific diseases is possible with these
probes.
[0081] The subject of the present invention is also a sequence
segment of a treated DNA which is at least 18 bases long according
to one of the [sequences] Seq. ID 1 to Seq. ID 40712. These
segments of 18 base pairs in length comprised of Seq. ID 1 to Seq.
ID 40712 are utilized for the amplification of the treated genomic
DNA. Oligomers with a length of at least 9 nucleotides are used as
detectors of these segments.
[0082] The oligomers preferably contain at least one CpG
dinucleotide. The cytosine of the corresponding CpG dinucleotide is
found in approximately the middle third of the oligomer. It is a
deciding factor that at least one oligonucleotide from Seq. ID 1 to
Seq. ID 40712 is present in the respective set of oligomers for at
least each of the CpG dinucleotides.
[0083] The oligomers are preferably produced on a support material
in a fixed arrangement, whereby at least one oligomer is coupled to
a solid phase. Methods for binding oligomer probes to solid phases
are known to the person of average skill in the art.
[0084] It is also important in this connection that it is not
individual CpG dinucleotides, but the large number of CpG
dinucleotides present in the sequences, which must be analyzed for
the diagnosis of genetic and/or epigenetic parameters of the
claimed nucleic acids (Seq. ID 1 to Seq. ID 40712). In a
particularly preferred variant of the method, all of the CpG
dinucleotides present in the sequences are to be investigated.
[0085] It is further preferred that all oligomer probes have the
same length. In addition, all 18-mer [segments] which have a CpG
dinucleotide in the center and which hybridize to one of the Seq.
ID 1 to Seq. ID 40712 without erroneous base pairing are
particularly preferred.
[0086] In another preferred variant of the method, at least ten of
the oligomers are used for the detection of the cytosine
methylation state and/or of single nucleotide polymorphisms (SNPS)
in chemically pretreated genomic DNA.
[0087] The oligomers are preferably used for the diagnosis of
undesired drug interactions; cancer diseases; CNS malfunctions,
damage or diseases; symptoms of aggression or behavioral
disturbances; clinical, psychological and social consequences of
brain lesions; psychotic disturbances and personality disorders;
dementia and/or associated syndromes; cardiovascular disease;
malfunction, damage or disorder of the gastrointestinal tract;
malfunction, damage or disorder of the respiratory system; lesion,
inflammation, infection, immunity and/or convalescence;
malfunction, damage or disorder of the body as an abnormality in
the development process; malfunction, damage or disorder of the
skin, the muscles, the connective tissue or the bones; endocrine
and metabolic malfunction, damage or disorder; headaches and sexual
malfunctions, by analysis of methylation patterns.
[0088] Also, of the nucleic acids or considerable segments thereof
listed in the sequence protocol (Seq. ID 1 to Seq. ID 40712),
preferably at least one will be used for the analysis of a set of
genetic and/or epigenetic parameters for the diagnosis of existing
disorders or for the diagnosis of predisposition for specific
disorders.
[0089] The person of average skill in the art understands that the
oligomers fulfill the same objective when thymine is exchanged for
uracil.
[0090] The genomic DNA to be analyzed is obtained preferably from
the usual sources for DNA, such as, e.g., cell lines, blood,
sputum, stool, urine, cerebrospinal fluid, tissue embedded in
paraffin, for example, tissue from eyes, intestine, kidney, brain,
heart, prostate, lung, breast or liver, histological slides and all
other possible combinations thereof.
[0091] The subject of the present invention is also nucleic acids
containing a sequence segment which is at least 18 bases long of a
chemically pretreated DNA, according to one of the [sequences] Seq.
ID 1 to Seq. ID 40712.
[0092] The subject of the present invention is also an oligomer
(oligonucleotide or peptide nucleic acid (PNA) oligomer) for the
detection of the cytosine methylation state in chemically
pretreated DNA, each containing at least one base sequence with a
length of at least 9 nucleotides, which hybridizes to a chemically
pretreated DNA (Seq. ID 1 to Seq. ID 40712). It is also preferred
according to the invention that the base sequence contains at least
one dinucleotide. It is also preferred that the cytosine of the CpG
dinucleotide is found in approximately the middle third of the
oligomer.
[0093] The subject of the invention is also a set of oligomers
according to the invention, containing at least one oligomer for at
least one of the CpG dinucleotides of one of the sequences of Seq.
ID 1 to Seq. ID 40712. A set of oligomers containing at least one
oligomer for each of the CpG dinucleotides of one of the sequences
of Seq. ID 1 to Seq. ID 40712 is preferred.
[0094] The subject of the present invention is also a set of at
least two nucleic acids, which are utilized as primer
oligonucleotides for the amplification according to the invention
of at least one of the [sequences] Seq. ID 1 to Seq. ID 40712 or
segments thereof. It is preferred that at least one oligonucleotide
is bound to a solid phase.
[0095] The subject of the present invention is also a set of
oligomer probes for the detection of the cytosine methylation state
and/or of single nucleotide polymorphisms (SNPs) in chemically
pretreated genomic DNA according to one of the [sequences] Seq. ID
1 to Seq. ID 40712, containing at least ten of the above-named
oligomers according to the invention.
[0096] The subject of the present invention is also a method for
the production of an arrangement of different oligomers (an array)
fixed on a support material for the analysis of disorders related
to the methylation state of the CpG dinucleotides of one of the
[sequences] Seq. ID 1 to Seq. ID 40712, in which at least one
oligomer according to the invention is coupled to a solid
phase.
[0097] The subject of the invention is also arrangements of
different oligomers (array) bound to a solid phase.
[0098] The subject of the present invention is also an array of
different oligonucleotide and/or PNA oligomer sequences whereby
these are arranged on a planar solid phase in the form of a
rectangular or hexagonal grid. It is preferred that the solid phase
surface is comprised of silicon, glass, polystyrene, aluminum,
steel, iron, copper, nickel, silver, or gold.
[0099] According to the invention, a DNA and/or PNA array is also
[included] for the analysis of disorders related to the methylation
state of genes, which contains at least one nucleic acid as
described above according to the invention.
[0100] The following examples explain the invention.
EXAMPLE 1
Production of Unmethylated and Methylated DNA and Bisulfite
Treatment
[0101] For the production of methylated DNA, human genomic DNA was
treated with S-adenosylmethionine und CpG methylase (Sssl, New
England Biolabs,) according to the information of the manufacturer.
For the production of unmethylated DNA, the gene fragment ELK-1
(Accession number ep59011) was amplified by means of PCR with the
primers GCTCTATGGTCTTGTCTAACCGTA and AGGTGGTGGTGGCGGTGG, starting
from human genomic DNA. The unmethylated and methylated DNA, which
was prepared in this way, as well as also the human genomic DNA was
treated with the use of bisulfite (hydrogen sulfite, disulfite),
such that all cytosines unmethylated at the 5-position of the base
are changed so that a base that is different with respect to base
pairing behavior is formed, whereas the cytosines that are
methylated in the 5-position remain unchanged. If bisulfite in the
concentration range between 0.1 M and 6 M is used for the reaction,
then an addition occurs at the unmethylated cytosine bases. Also, a
denaturing reagent or solvent as well as a radical trap must be
present. A subsequent alkaline hydrolysis then leads to the
conversion of unmethylated cytosine nucleobases to uracil. This
converted DNA serves for the detection of methylated cytosines.
EXAMPLE 2
Production of Cy5-labeled Gene Probes
[0102] Starting with DNA samples treated with bisulfite, a defined
fragment of 529 bp in length from the promoter region of the ELK-1
gene (Accession number ep59011) was amplified. The amplification is
conducted with the primer oligonucleotides
ATGGTTTTGTTTAATYGTAGAGTTGTTT and TAAACCCRAAAAAAAAAAACCCAATAT. By
using primer oligonucleotides that are labeled with the fluorescent
dye Cy5, the fragment is directly labeled in the PCR. (1)
Unmethylated DNA, (2) methylated DNA and (3) human genomic DNA
treated with bisulfite (hydrogen sulfite, disulfite) are used as
the matrix DNA. Then these three different DNA fragments are
investigated in separate hybridizations for their degree of
methylation at a specific CpG position.
EXAMPLE 3
Conducting the Hybridization and Evaluating a Hybridized DNA
Chip
[0103] The gene probes prepared in Example 2 are hybridized to a
DNA chip. First, oligonucleotides are immobilized on the chip. The
oligonucleotide sequences are derived from the amplified fragment
of the ELK-1 gene named in Example 2, and represent the CG
dinucleotide, including the immediate surroundings. The length of
the oligonucleotides amounts to 14-22 nucleotides; the position of
the CG dinucleotide within the oligonucleotide is variable. After
the hybridization, the DNA chip is scanned (see FIG. 1) and the
hybridization signals are numerically evaluated (data not shown).
The result of the hybridization for the oligonucleotides
CTACTCAACGAAAACAAA and CTACTCAACAAAAACAAA is shown in FIG. 1 and
FIG. 2. CTACTCAACGAAAACAAA preferably hybridizes if the cytosine of
the ELK-1 fragment, which is found at position 103 of the amplified
product, is methylated; CTACTCAACAAAAACAAA hybridizes if this
cytosine is unmethylated.
[0104] A DNA chip is shown in FIG. 1 after hybridization with the
ELK-1 fragment. The pseudo-color image as it is produced after
scanning is shown. Unlike the black-and-white illustration shown
here, a color image is produced by the scanner. The intensity of
the different colors represent the degree of hybridization, whereby
the degree of hybridization decreases from red (this can be
recognized as light spots in FIG. 1) to blue (recognized as dark
spots in FIG. 1).
[0105] FIG. 2A shows an excerpted image from FIG. 1. The spotted
pairs of oligonucleotides are circled in white to clarify the
hybridization diagram: ctactcaacaaaaacaaa (left) and
ctactcaacgaaaacaaa (right).
[0106] The excerpted image of FIG. 2B shows the spotting pattern
for an unknown methylation state of the tissue sample and the
excerpted image of FIG. 2C shows the methylation state for the
methylated reference sample.
1 TABLE 1 Sample A Sample B Sample C (unmethylated) (unknown)
(methylated) Sequence of the Fluorescence Fluorescence Fluorescence
detection oligomer (counts) Mean (counts) Mean (counts) Mean
ctactcaacgaaaacaaa 3352 6102 6002 ctactcaacgaaaacaaa 2950 6775 7898
ctactcaacgaaaacaaa 4196 6360 7485 ctactcaacgaaaacaaa 5181 5521
11401 3920 6190 8197 ctactcaacaaaaacaaa 20577 7074 7290
ctactcaacaaaaacaaa 19709 9171 9985 ctactcaacaaaaacaaa 24130 7603
9286 ctactcaacaaaaacaaa 21601 9434 12435 21504 8321 9749 CG/CA 0.28
0.74 0.84
[0107] The mean is indicated each time for a wavelength of 635 nm.
Column A gives the values for the unmethylated [reference] sample
and column B for an unknown methylation state of a tissue sample
and column C gives the values for the methylated reference sample.
The CG/CA ratios represent the methylation state of the respective
sample. The value of 0.74 shows that the sample is basically
present in methylated form.
EXAMPLE 4
[0108] The following example relates to a fragment of the hMLH1
gene associated with hereditable non-polyposis colorectal cancer,
in which a specific CG position is investigated for
methylation.
[0109] In the first step, a genomic sequence is treated with the
use of bisulfite (hydrogen sulfite, disulfite) in such a way that
all of the unmethylated cytosines at the 5-position of the base are
modified such that a base that is different in its base pairing
behavior is formed, while the cytosines that are methylated in the
5-position remain unchanged. If bisulfite in the concentration
range between 0.1 M and 6 M is used for the reaction, then an
addition occurs at the unmethylated cytosine bases. Also, a
denaturing reagent or solvent as well as a radical trap must be
present. A subsequent alkaline hydrolysis then leads to the
conversion of unmethylated cytosine nucleobases to uracil. This
converted DNA serves for the detection of methylated cytosines. In
the second step of the method, the treated DNA sample is diluted
with water or an aqueous solution. A desulfonation of the DNA
(10-30 min, 90-100.degree. C.) at alkaline pH is then preferably
conducted. In the third step of the method, the DNA sample is
amplified in a polymerase chain reaction, preferably with a
heat-stable DNA polymerase. In the present Example, cytosines of
the hMLH1 gene, here from a 1551-bp-long 5'-flanking region, are
investigated. A defined fragment of 719-bp length is amplified for
this purpose with the specific primer oligonucleotides
AGCAACACCTCCATGCACTG and TTGATTGGACAGCTTGAATGC. This amplified
product serves as a sample, which hybridizes to an oligonucleotide
that has been previously bound to a solid phase, with the formation
of a duplex structure, for example, GAAGAGCGGACAG, whereby the
cytosine to be detected is found at position 588 of the amplified
product. The detection of the hybridization product is based on
primer oligonucleotides fluorescently labeled with Cy3 and Cy5,
which were used for the amplification. A hybridization reaction of
the amplified DNA with the oligonucleotide occurs only if a
methylated cytosine was present at this site in the
bisulfite-treated DNA. Thus the methylation state of the respective
cytosine to be investigated decides the hybridization product.
EXAMPLE 5
Production of Bisulfite-Modified DNA with Agarose Beads
[0110] In the present experiment, starting with DNA treated with
bisulfite, a defined fragment of 529 bp in length from the promoter
region of the ELK-1 gene (Accession number ep59011) is amplified.
By using primer oligonucleotides ATGGTTTTGTTTAATYGTAGAGTTGTTT and
TAAACCCRAAAAAAAAAAACCCAATAT, which are labeled with the fluorescent
dye ALEXA 488, the fragment is directly labeled in the PCR.
Oligonucleotides of the first class (here, for example,
ATTAATAGCGTTTTGGTT) and of the second class (here: for example,
ATTAATAGTGTTTTGGTT) are immobilized at the surface of beads, which
are distinguished by an individual color coding. In a following
step, the amplified products of the ELK-1 gene, which were prepared
with the above-named primer oligonukleotides, are combined with a
mixture of both classes of beads, whereby the amplified products
hybridize to the immobilized oligonucleotides, here, for example,
ATTAATAGCGTTTTGGTT and ATTAATAGTGTTTTGGTT, whereby the C or T to be
detected is found each time at position 476 of the amplified
product. Then the beads are separated, identified by fluorescence
measurement based on their color coding and the degree of
hybridization is determined by measurement of the fluorescent
intensities, which are specific to the fluorescent dye ALEXA
488.
EXAMPLE 6
Use of Multiple Dyes for Internal Calibration
[0111] Starting with bisulfite-treated DNA, a defined fragment of
529-bp length from the promoter region of the ELK-1 gene is
amplified. The fragment is labeled directly in the PCR by use of
the fluorescently labeled primer oligonucleotides
ATGGTTTTGTTTAATYGTAGAGTTGTTT and TAAACCCRAAAAAAAAAAACCCAATAT. For
the PCR reaction, which is conducted on a thermocycler (Eppendorf
GmbH), 10 ng of bisulfite-treated DNA, 6 pmol of each primer, 200
.mu.M of each dNTP, 1.5 mM MgC12 and 1 U of HotstartTaq (Qiagen AG)
are used. The other conditions are selected following the
manufacturer's information. For the amplification, a denaturing is
conducted for 14 min at 96.degree. C., followed by 39 cycles with
the conditions: 60 sec at 96.degree. C, 45 sec at 55.degree. C. and
75 sec at 72.degree. C. In conclusion, an elongation is conducted
for 10 min at 72.degree. C. In the present case, the samples to be
investigated, here the tissue of healthy and sick persons, are
labeled with the Cy2 dye. For the internal calibration of the
methylation state, primer oligonucleotides are used for the
amplification of samples for the calibration, which are labeled
with the fluorescent dyes Cy3 and Cy5. The amplified products from
the samples for the calibration represent a known methylation
state, on the one hand, a state of one-hundred percent methylation,
and, on the other hand, an unmethylated state. Since in this
method, the ratios of the color intensities of the fluoresent dyes,
which are measured with the use of the ScanArray 4000XL, Packard
BioScience-BioChip Technologies, are calculated for two classes of
oligonucleotides, here, for example, ATTAATAGCGTTTTGGTT and
ATTAATAGTGTTTTGGTT, wherein the C or T to be detected is found each
time at position 476 of the amplified product, the ratio of
methylated to unmethylated state of the unknown sample can be
determined.
EXAMPLE 7
Use of Multiple Dyes for Increasing the Sample Throughput Volume
and for Increasing the Complexity of the Analysis
[0112] The present experiment serves for the purpose of analyzing
different samples in a single hybridization step and in this way
increasing the sample throughput volume. For this purpose, a
defined fragment of 529-bp length from the promoter region of the
ELK-1 gene taken from each of four individuals and, starting with
the bisulfite-treated DNA, is amplified with the primer
oligonucleotides ATGGTTTTGTTTAATYGTAGAGTTGTTT and
TAAACCCRAAAAACCCAATAT. These fragments originating from four
individuals are labeled with the four different fluorescent dyes
Cy3, Cy5, Cy2 und Cy7 and hybridized to immobilized
oligonucleotides, here, for example, ATTAATAGCGTTTTGGTT and
ATTAATAGTGTTTTGGTT, whereby the C or T to be detected is found each
time at position 476 of the amplified product. The samples with
different fluorescent labels are then analyzed at different
wavelengths without a mutual interference based on the fluorescent
dye.
[0113] On the other hand, it is also possible to produce different
sets of fragments from one DNA sample, which [fragments] are
labeled with different dyes, here Cy2, Cy3 and Cy5. If a set of
oligonucleotide probes for 64 genes (set 1) is immobilized on a
chip, then the specificity is sufficient in order to analyze 64
fragments, e.g., labeled with Cy3, independent of one another. Due
to the fact that samples of set 1 are labeled here with the
fluorescent dye Cy3 and samples of set 2 are labeled with the
fluorescent dye Cy5 (and set 3 with Cy2), the detection of the
methylation state via measuring the fluoresent intensities of the
fragments of set 1 is not influenced by the fluorescently labeled
amplified products of sets 2 and 3 (and vice versa). Therefore,
despite the increased complexity of the amplified products, it is
possible to produce data that are equally reliable to those for a
[lesser] complexity of 64 amplified products.
EXAMPLE 8
Use of Multiple Dyes for Verifying Experimental Reproducibility
[0114] In the present experiment, the same PCR amplified products
are labeled with four different fluorescent dyes and these are
verified [for reliability] by a 4.times. redundancy. For this
purpose, starting from bisulfite-treated DNA, a a defined fragment
of 529-bp length from the promoter region of the ELK-1 gene is
amplified with the primer oligonucleotides
ATGGTTTTGTTTAATYGTAGAGTTGTTT and TAAACCCRAAAAAAAAAAACCCA- ATAT, and
hybridized to immobilized oligonucleotides, here, for example,
ATTAATAGTGTTTTGGTT und ATTAATAGTGTTTTGGTT, whereby the C or T to be
detected is found each time at position 476 of the amplified
product. The ratios of samples with different fluorescent labels
are compared by employing primer oligonucleotides fluorescently
labeled with Cy3, Cy5, Cy2 and Cy7, and in this way a higher
experimental reliability is achieved.
Sequence CWU 0
0
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