WO2014020124A1 - Moyens et procédés pour la détection d'une méthylation de l'adn - Google Patents
Moyens et procédés pour la détection d'une méthylation de l'adn Download PDFInfo
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- WO2014020124A1 WO2014020124A1 PCT/EP2013/066221 EP2013066221W WO2014020124A1 WO 2014020124 A1 WO2014020124 A1 WO 2014020124A1 EP 2013066221 W EP2013066221 W EP 2013066221W WO 2014020124 A1 WO2014020124 A1 WO 2014020124A1
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- 0 CCCCC(C)CC(C)(C(*(C)[N+](C(*C*)*=I)[O-])O)C(*)(*)C(*)OC Chemical compound CCCCC(C)CC(C)(C(*(C)[N+](C(*C*)*=I)[O-])O)C(*)(*)C(*)OC 0.000 description 2
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6846—Common amplification features
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- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/161—Modifications characterised by incorporating target specific and non-target specific sites
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- C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
- C12Q2535/125—Allele specific primer extension
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- C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
- C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
- C12Q2537/164—Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
Definitions
- the present invention relates to the means and methods for the detection of the methylation status of cytosine residues and in particular to the use of at least one primer which is at least 5 nucleotides in length, and which hybridizes to a target sequence that comprises at least one defined 5 ' -CpG-3 ' which target sequence is comprised in a genomic DNA of a vertebrate, and which has its 3 " end opposite to the C of said 5 ' -CpG-3 ' , and which comprises at said 3 " end a mismatched nucleotide in relation to said C of said 5 ' -CpG-3 ' , for determining the methylation status of a vertebrate genomic nucleic acid at said C.
- the present invention further relates to polymerase proteins that can be used in the embodiments of the present invention.
- the present invention also relates to a kit comprising at least one primer of the invention and optionally means to conduct the amplification.
- the present invention further relates to a method for directly detecting methylation or hydroxymethylation of a cytosine residue of interest in a DNA molecule.
- Said method comprises steps of providing a primer having its 3'-end opposite of the cytosine residue of interest and having at said 3'-end a mismatched base, performing a specific DNA polymerase reaction, such as primer extension, rolling circle amplification (RCA) or polymerase chain reaction (PCR), with said primer using said DNA molecule as template, and detecting said methylation or hydroxymethylation via an increased efficiency of said specific DNA polymerase reaction as compared to the same reaction performed with an unmodified DNA molecule as template.
- a specific DNA polymerase reaction such as primer extension, rolling circle amplification (RCA) or polymerase chain reaction (PCR)
- Different cells of an organism display broad functional and morphological diversity, although they all possess the same genetic material. Differential gene expression is the cause for this heterogeneity.
- the term "epigenetics" relates to all research in this field. It is defined as the study of inheritable, phenotypical changes in the gene expression pattern of a specific cell type that are not caused by a transformed nucleotide sequence of the genetic code itself.
- a coding for the gene expression state which was postulated for the first time 36 years ago, is flexible enough to support specialization of genetically identical somatic cells towards different functions and to enable reactions to regulatory impacts from other cells or from external stimuli. Further, this coding is stable enough to persist in the germ cells and to be passed from one generation to the next.
- Epigenetic markers are represented by a variety of molecular mechanisms, such as posttranslational histone modifications, ATP-dependent chromatin remodeling, small and other non-coding RNA (siRNA, miRNA), binding of histone variants and non-histone proteins, polycomb-trithorax protein complexes and last but not least DNA methylation and hydroxymethylation.
- siRNA small and other non-coding RNA
- miRNA binding of histone variants and non-histone proteins
- polycomb-trithorax protein complexes last but not least DNA methylation and hydroxymethylation.
- 5-Hydroxymethylcytosine (Fig. 1 C) was first discovered in the bacteriophages T2, T4 and T6 in 1952. The presence of it in mammalian DNA was suggested not until twenty years later, but has received only little scientific attention. In 2009, 5-hydroxymethylcytosine was detected in cerebellar Purkinje neurons in the brain, where it constitutes 0.6% and 0.2% of all bases in Purkinje cells and granule cells, respectively. Simultaneously, 5- hydroxymethylcytosine was reported to be present in mouse embryonic stem cells and human embryonic kidney cells.
- the TET1 (ten-eleven translocation 1 ) protein a fusion partner of histone methyltransferase in acute myeloid leukemia, was identified as a 2-oxoglutarate- and Fe(l Independent enzyme that catalyzes the conversion of 5-methylcytosine to 5- hydroxymethylcytosine in vitro, as well as in cultured cells.
- the three paralogous human proteins TET1 , TET2 and TET3 were found as they have homologous regions to the oxygenase domains of JBP1 and JBP2 that are known to catalyze the initial step of base J ( ⁇ - D-glucosyl hydroxymethyluracil) biosynthesis in trypanosomes.
- 5- hydroxymethylcytosine could be an intermediate in the pathway of an active demethylation, as active methylation has been observed during different steps of development.
- the responsible enzymes have been elusive.
- two studies showed that 5-methylcytosine as well as 5-hydroxymethylcytosine are oxidized to 5-formylcytosine and 5-carboxylcytosine by Tet dioxygenases in cultured cells and in vitro, and that thymine-DNA glycosylases specifically recognize and excise 5-carboxylcytosine as a part of base excision repair.
- 6-Cytosine-sulphonate is spontaneously deaminated in aqueous solution. Ammonium is formed as a by-product. Then, NaOH, which is added again, leads to cleavage of uracil sulphonate into uracil and bisulphite. After the conversion reaction by bisulphite, the DNA sample is amplified by PCR. Two strategies are possible. First, two primer pairs are chosen which span the 5 ' -CpG-3 ' site. Hereby, one primer pair is designed for unmethylated DNA and the other primer pair for methylated DNA. This is called methylation specific PCR. Second, only one primer pair is used which flanks a 5 ' -CpG-3 ' site.
- each 5-methylated cytosine is replaced by an unmethylated cytosine and each uracil is replaced by thymine.
- 5-Hydroxymethylcytosine reacts with bisulphite to yield cytosine-5-methylenesulfonate which does not promote deamination and therefore, also codes as cytosine.
- sodium bisulphite treatment does not distinguish between 5-methylcytosine and 5-hydroxymethylcytosine.
- bisulphite sequencing has many disadvantages. Bisulphite sequencing uses very harsh chemicals and can cause DNA fragmentation. Due to the bisulphite conversion, the sequence, if unmethylated, is reduced to only three nucleotides (A, G, T(U)). This complicates the primer design and alignments to the reference sequence. Furthermore, two types of bisulphite conversion errors can occur: either an inappropriate conversion of 5-methylcytosine to thymine or the failure to convert unmethylated cytosine to uracil. The frequency of the error that is mentioned first was found to range from 0.09 to 6.1 % for selected protocols.
- the technical problem underlying the present invention is to provide means and methods for the detection of the methylation status of a DNA molecule.
- the primer provided by the present invention is preferably designed such that its outmost 3'-end is opposite to the cytosine residue of interest (i.e. the one whose methylation status should be analyzed) and has a mismatched base in respect to the cytosine of interest. Said mismatched base at the 3'-end of the primer does not sufficiently pair with the cytosine of interest, in case said cytosine is not methylated or hydroxymethylated, thus impairing for example a DNA polymerase reaction with said primer using said DNA molecule as template.
- said mismatched base at the 3'-end of the primer pairs sufficiently with the cytosine of interest, in case said cytosine is methylated or hydroxymethylated, thus allowing a DNA polymerase reaction with said primer using said DNA molecule as template (see for example Fig. 2 for further illustration).
- the readout of said method is straightforward as it is for example merely necessary to analyze the influence of the methylation status at the cytosine residue of interest on the downstream extension reaction (see the appended examples and Figures 3 and 4 for illustration).
- the uses and methods of the present invention are significantly less time-, labor- and cost-intensive compared to methods known in the art. Moreover, they are much less prone to errors and allow the analysis of very small amounts of sample material.
- the present invention thus relates in essence to the use of at least one primer:
- a “primer” is an oligonucleotide, typically between about 5 to 200 nucleotides in length, capable of selectively binding to a specified nucleic acid (the target sequence) by hybridizing with the target sequence.
- the primer of the invention are generally made of less than 1 ,000 nucleotide (nt), including those in a size range having a lower limit of about 5 nt and an upper limit of about 500 to 900 nt.
- Preferred primer are in a size range having a 5 to 15 nt lower limit and a 50 to 500 nt upper limit, and particularly preferred embodiments are in a size range having a 10 to 20 nt lower limit and a 25 to 150 nt upper limit.
- oligonucleotide includes DNA and/or RNA or modifications thereof, comprising nucleotides such as e.g. the conventional bases (A, G, C, T, U) and/or nucleotide analogs as monomeric units.
- An "analog” or “nucleotide analog” can refer to a nucleotide-like molecule such as a structural moiety that can act substantially like a nucleotide, for example exhibiting base complementarity with one or more of the bases that occur in DNA or RNA and/or being capable of base-complementary incorporation via an enzymatic reaction (e.g.
- Nucleotide analogs comprise in one example nitrogenous heterocyclic bases (e.g., inosin).
- the above mentioned monomeric units are typically covalently linked by standard phosphodiester bonds or other linkages.
- the backbone may be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid (PNA) linkages, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof to name some.
- Locked Nucleic Acid (LNATM) nucleosides analogues are also envisaged.
- the primers of the present invention can be modified and/or labeled, i.e. they may further include non-nucleotide groups such as, for example, abasic nucleotides, universal bases (e.g., 3-nitropyrrole and 5-nitroindole), polysaccharides, peptides, polypeptides etc.
- non-nucleotide groups such as, for example, abasic nucleotides, universal bases (e.g., 3-nitropyrrole and 5-nitroindole), polysaccharides, peptides, polypeptides etc.
- label is meant a reporter moiety associated with a primer which can be detected by means well known in the art and used to indicate the presence or absence of a particular polynucleotide sequence in a test sample.
- labels which are well known in the art include chemiluminescent, electrochemiluminescent and fluorescent compounds, radioisotopes, dyes, polynucleotides, enzymes, enzyme substrates, chromophores and haptens etc.
- interacting labels may include, for example, the following: luminescent and quencher labels, luminescent and adduct labels, dye dimer labels, enzyme and substrate labels, enzyme and cofactor labels, and Forrester energy transfer pairs.
- Radioactive P end-labeled primer (particularily 5 " -
- P end labeled primer are less preferred.
- one, two, three, or four types of nucleotides are differentially labeled.
- four different types of nucleotides are labeled with four different labels.
- the primer of the invention may also include a "tag" sequence, which may be used to identify the primer, for example, to distinguish the primer from other, similar primer. Any nucleic acid analog is contemplated by the primers of the present invention, provided the primer can form a stable hybrid with the target sequence under hybridization conditions and, provided the primer can be extended from its 3 ' -end.
- “Hybridizing” refers to the ability to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
- hybridization conditions or “hybridizing” or “hybridize” or grammatical variants thereof are meant conditions permitting a primer to stably hybridize to a target sequence (the complex is then e.g. stabilized via hydrogen bonding between the bases of the nucleotide residues) which is a prerequisite for initiation of template-directed synthesis of a nucleic acid complementary to the target sequence.
- Hybridization conditions may vary depending upon factors including the GC (guanine/cytosine) content and length of the primer, the degree of similarity between the primer sequence and sequences of non-target nucleic acids which may be present in the test sample, and the target sequence.
- Hybridization conditions include the temperature and composition of the hybridization reagents or solutions.
- the hybridization is performed under conditions in which the target sequence to be probed is single-stranded.
- the primer of the present invention has its 3 " end opposite to the C of said 5 ' -CpG-3 ' (see Fig. 2B or Fig. 3A).
- the primer of the invention also provides a point of initiation for polymerase-mediated template-directed synthesis of a nucleic acid complementary to the template sequence.
- the "template sequence” is usually directly downstream to the target sequence.
- “Template” refers to a single-stranded nucleic acid, or a denatured region of a double-stranded nucleic acid, that a polymerase can utilize to synthesize a complementary nucleic acid strand.
- a primer is thus capable of being extended from its 3 " -end, i.e. the 3'-end of the primer provides a free 3'-OH group whereto further "nucleotides” may be attached by a template- dependent DNA polymerase.
- "Being extended” or “extended from its 3 " -end” or “extending ...from its 3 " -end” etc. thereby includes typical "nucleic acid amplification” -methodology in all variations.
- nucleic acid amplification uses one or more nucleic acid polymerase and/or transcriptase enzymes to produce multiple copies of a target sequence or fragments thereof, and/or of a sequence complementary to the target sequence or fragments thereof.
- TMA transcription mediated amplification
- NASBA nucleic acid sequence based amplification
- PCR Polymerase Chain Reaction
- qRT-PCR quantitative real-time PCR
- qRT-PCR quantitative real-time PCR
- reverse transcriptase-PCR reverse transcriptase-PCR
- LCR Ligase Chain Reaction
- the primer of the present invention comprises at its 3 " end a mismatched nucleotide in relation to said C of said 5 ' -CpG-3 ' .
- the present inventors discovered that the methylation or hydroxymethylation of the "C" of said at least one defined 5 ' -CpG-3 ' influences the extension reaction from said 3 ' -end when using the primer of the present invention.
- the rationale is as follows, without being bound by theory.
- the primer of the present invention has its 3 " end opposite to the C of said 5 ' -CpG-3 ' , and comprises at said 3 " end a mismatched nucleotide in relation to said C of said 5 ' -CpG-3 ' .
- a “mismatched" nucleotide or base (or nucleoside etc.) in relation to said "C” thereby means in essence that the very mismatched nucleotide etc. is not complementary to said "C” thereby excluding in a preferred embodiment a "G" at the utmost 3 ' -end of the primer. It is thus preferred that the mismatched nucleotide etc. is selected from the group consisting of adenine, cytosine, thymine, inosine, uracil (A, C, T, I, U) or modifications thereof. It is envisaged that said mismatched nucleotide etc.
- the primer does not canonically pair with the cytosine of interest, in case said cytosine is not methylated or hydroxymethylated, which negatively impairs the 3 ' -extension reaction of said primer.
- the methylation/hydroxymethylation of said "C” somehow compensates for said mismatch which positively impairs the 3 ' -extension reaction of said primer (see for example Fig. 2 for further illustration).
- the methylation/hydroxymethylation status of the "C" in the at least one 5 ' -CpG-3 ' thus influences the extension reaction from the 3 " -end of the primer of the invention.
- Primers hybridizing to opposing strands of a double- stranded target sequence are referred to as forward and reverse primers.
- the primer is substantially complementary to its target sequence.
- substantially complementary in reference to two nucleic acids, means that the two nucleic acids each contain hybridization regions that are of sufficiently complementary as to be able to interact with each other in a specific, determinable fashion, i.e., when the two nucleic acids are brought together in an antiparallel orientation, the same nucleotides of each nucleic acid will become hybridized to each other at one or more specific locations.
- “Substantially complementary” also means that the primer is almost completely or partially complementary to its target sequence.
- Partially complementary encompasses primer that are at least 70%, 75%, 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 96, 97, 98 or 99% complementary to their target sequence.
- sequence identity a comparison is made by aligning the sequences in a manner to provide the maximum correspondence of nucleotides. It is envisaged, however, that the primers of the present invention are in a preferred embodiment not 100% complementary to their target sequence because at least the 3 " -end of the primer of the invention is mismatched in relation to the C of the 5 ' -CpG-3 ' , as described herein elsewhere.
- the two hybridization regions may have a maximum of 30 mismatches, 20 mismatches, 10 mismatches, or 7 mismatches. In still other cases, the two hybridization regions may have a maximum of 6, 5, 4, 3, 2, or 1 , mismatches.
- the primer further comprises the base C (e.g. contained in a nucleotide or nucleoside) at the penultimate position in relation to its 3 " end.
- the base C e.g. contained in a nucleotide or nucleoside
- Determining the methylation status of a vertebrate genomic nucleic acid at said “C” means the “C” that is comprised in a 5 ' -CpG-3 ' site.
- the "methylation status” thereby denotes the presence or absence of a methyl-residue or hydroxymethyl-residue at said "C", and in particular at the C5-atom of said "C” (see Fig. 1 for illustration). It is envisaged that "determining the methylation status” comprises a qualitative detection (presence or absence) and/or a quantitative or semi-quantitative detection of the methylation/hydroxymethylation status.
- determining means determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute.
- Cytosine methylation is crucial for mammalian embryogenesis. During this process, methylation levels change dynamically. There are various cell-type specific epigenomes with a well-defined methylation pattern which occurs in differentiation of the mammalian organism. Differentiation is characterized by two waves of genome-wide epigenetic reprogramming in the zygote and in the primordial germ cells. The genome becomes demethylated during preimplantation in mice. The maternal genome remains methylated or undergoes de novo methylation, whereas the paternal genome is rapidly and actively demethylated. Through cell divisions, the loss of maternal methylation markers occurs passively until blastocyst formation. In implementation, when the cell lines start to develop to different lineages, the methylation level is restored de novo.
- DNA methyltransferases which are responsible for the methylation of cytosines, are essential and a dysfunction in any of them leads to embryonic lethality.
- the second wave only occurs in the primordial germ cells where DNA methylation patterns are deleted at all single-copy genes.
- Ageing and cellular senescence are also characterized by a decrease of the overall content of DNA methylations.
- specific sites of distinct genes acquire methylation, for example at their promoters. This situation is similar to methylation changes in cancer or other diseases.
- a hypomethylated promoter leads to active gene expression, whereas a gene with a hypermethylated promoter is silenced. It is supposed that 5 ' -CpG-3 ' methylation directly disturbs the binding of transcriptional regulators to their appropriate DNA sequences. Another possibility could be the recruitment of methyl-5 ' -CpG-3 ' binding proteins which leads to a repressed chromatin environment.
- DNA methylation is closely interconnected with chromatin remodeling and histone modification.
- 5-methylcytosines In somatic cells, about 1 % of DNA bases are 5-methylcytosines. The abundance of 5- methylcytosine varies slightly in different tissue types. 5-Methylcytosines are frequently found as symmetrical 5-methylations of the dinucleotide CpG within or nearby promoters. Here, 75% of them are methylated throughout the mammalian genomes. CpG dinucleotides are underrepresented in the genome since they are mutation hotspots. Methylated CpGs can be deaminated to the naturally occurring DNA bases TpGs which cannot be repaired. Therefore, mutation rates of CpG sites are about 10 to 50 times higher than other transitional mutations and have led to depletion of the dinucleotide during evolution.
- CpG-rich clusters of a length of one to four kilobases are observed in promoter regions and the first exon of various genes. They are called CpG islands of which there are about 30,000 in the human genome. CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates. 88% of active promoters are associated with CpG-rich sequences and might be regulated by DNA methylation. Their susceptibility to become methylated alters during development and carcinogenesis. [0018] It is also envisaged that the embodiments according to the invention are used in diagnostics, for diagnostic analysis or for bioanalytics, or for the screening of tissue or fluids from the human or even animal body for the presence of certain methylation pattern. Other possible uses are disclosed herein elsewhere.
- target sequence denotes said part of vertebrate genomic nucleic acid that comprises at least one defined 5 ' -CpG-3 ' which is the 5 ' -CpG-3 ' of interest and represents the sequence to which the primer hybridizes to.
- the target sequence is or is comprised in a vertebrate genomic nucleic acid.
- Said vertebrate genomic nucleic acid/DNA or “genomic nucleic acid/DNA of a vertebrate” is either isolated (for example extracted from a vertebrate by standard methods) or still part of an isolated biological sample of said vertebrate (e.g. a cell sample, such as a blood and/or cancer cell etc.; a body fluid, such as blood, serum, liquor, cerebrospinal fluid, amniotic fluid, peritoneal fluid, interstitial fluid, excretions (urine, stool) etc.; and/or or tissue such as biopsy material, cancer tissue etc.).
- a cell sample such as a blood and/or cancer cell etc.
- body fluid such as blood, serum, liquor, cerebrospinal fluid, amniotic fluid, peritoneal fluid, interstitial fluid, excretions (urine, stool) etc.
- tissue such as biopsy material, cancer tissue etc.
- the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate.
- the tissue sample may also be primary or cultured cells or cell lines.
- the tissue or cell sample is obtained from a disease tissue/organ.
- said vertebrate genomic nucleic acid is a mammalian genomic nucleic acid.
- said mammalian genomic nucleic acid is human genomic nucleic acid (including mitochondrial DNA).
- the vertebrate genomic DNA may comprise the whole genome or only parts thereof (such as single chromosomes, mitochondrial DNA), or fragments thereof (e.g. fragments of a particular size range, generally of about 200-600 base pairs).
- the vertebrate genomic DNA is physically fragmented for example sonicated, sheared, or enzymatically fragmented etc. It is further envisaged that the vertebrate genomic DNA is at least 35nt in length. It is also envisaged that the genomic DNA is single or double stranded.
- genomic DNA in particular the fragments
- the genomic DNA are immobilized to a solid support.
- multiple genomic DNA fragments may be separated and analyzed by immobilizing the genomic DNA fragments on discrete areas of a solid support, e.g., on a microarray.
- genomic DNA fragments of interest may be immobilized and anaylzed in a high throughput manner.
- the solid support can have a variety of configurations, e.g., including, but not limited to, planar supports, non-planar supports, a sheet, bead, particle, slide, wafer, web, fiber, tube, capillary, microfluidic channel or reservoir, or other structure.
- the solid support may be porous or non- porous.
- the substrate may be formed from any suitable material, depending upon the application.
- the substrate may be a silicon-based chip or a glass slide.
- suitable substrate materials for the arrays of the present invention include, but are not limited to, glasses, ceramics, plastics, metals, alloys, carbon, agarose, silica, quartz, cellulose, polyacrylamide, polyamide, polyimide, and gelatin, as well as other polymer supports or other solid-material supports.
- a "microarray,” includes any one-dimensional, two-dimensional or substantially two-dimensional (as well as a three-dimensional) arrangement of addressable regions bearing a particular chemical moiety or moieties.
- vertebrate genomic nucleic acid comprises the target sequence and the template sequence that is employed in the context of the present invention.
- the target sequence of the present invention comprises at least one defined 5 ' -CpG-3 ' (sometimes also denoted as CpG site or 5 ' -CpG-3 ' or the like).
- CpG is shorthand for "— C— phosphate— G— ", that is, cytosine and guanine separated by only one phosphate.
- Said at least one 5 ' -CpG-3 ' can be comprised by a CpG island.
- a CpG island is usually a region comprised in a genomic DNA of a vertebrate, with at least 200 bp, and a GC percentage that is greater than 50%, and with an observed-to-expected CpG ratio that is greater than 60% (see for example Gardiner-Garden et al., Journal of Molecular Biology 196 (2): 261-82.).
- the "observed-to-expected CpG ratio" is calculated by formula ((Num of CpG/(Num of C ⁇ Num of G)) x Total number of nucleotides in the sequence)
- "Defined 5 ' -CpG-3"' means the 5 ' -CpG- 3 ' of interest.
- said target sequence is comprised by a gene depicted in the Tables herein (including the Table in Figure 5).
- said at least one defined 5 ' -CpG-3 ' , or the target sequence is comprised in a genetic element.
- the term "genetic element” includes for example regulatory elements such as a promoter, enhancer, silencer, insulator etc.; a gene or a part thereof such as an intron or exon; an intron/exon boundary, and/or a splicing region or other well-known genetic elements that can be identified in a genome of a vertebrate, such as transposons, etc.
- said at least one defined 5 ' -CpG-3 ' is comprised in a vertebrate genomic nucleic acid or fragment thereof and/or in a genetic element, wherein said vertebrate genomic nucleic acid or fragment thereof and/or said genetic element is associated with a disease (or disease state), such as for example a tumor suppressor gene, a genetic element controlling the expression of a tumor associated antigen etc. (exemplified herein).
- a disease or disease state
- diseases mentioned include but are not limited to neurological diseases, metabolic diseases, cardiovascular diseases, autoimmune diseases, cancer etc.
- said at least one defined 5 ' -CpG-3 ' is comprised in a vertebrate genomic nucleic acid or fragment thereof and/or in a genetic element, that is or is assumed to be differentially methylated in a disease.
- Such genetic elements or fragments of a vertebrate genomic nucleic acid
- a preferred selection comprises BRCA1 , MGMT PYCARD, RASSF1A, SHOX2 and/or SEPT9 but is not limited thereto.
- said vertebrate genomic nucleic acid, and/or the target sequence is not bisulfite treated.
- “Not bisulfite treated” means in essence that at least the target sequence that is employed in the context of the present invention, preferably the vertebrate genomic nucleic acid that is employed in the context of the present invention, is not suitable for/not prepared for bisulfite sequencing.
- “Bisulfite treated” or “Bisulfite treated” likewise refers to exposure of a nucleic acid to bisulfite ion (e.g., magnesium bisulfite or sodium bisulfite) at a concentration sufficient to convert unprotected cytosines to uracils.
- “Bisulfite treatment” also refers to exposure of a nucleic acid to other reagents that can be used to convert unprotected cytosines to uracils, e.g., disulfite and hydrogensulfite, at an appropriate concentration.
- “Bisulfite treatment” generally includes exposure of the nucleic acid to a base, e.g., NaOH, after exposure to the bisulfite ion or other reagent.
- Bisulfite sequencing is characterized by the selective chemical deamination of unmethylated cytosine to uracil by sodium bisulfite, as explained herein elsewhere.
- the present invention also relates to the use of a primer as defined herein in an oligonucleotide amplification method. It is envisaged that said oligonucleotide amplification method makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template.
- An oligonucleotide amplification method thereby denotes a method which is characterized by a step wherein the primer that is described herein is extended from its 3 " -end. "Extended from its 3 " -end” is explained herein elsewhere.
- the primer thus provides a point of initiation for polymerase-mediated template-directed synthesis of a nucleic acid complementary to the template sequence.
- Oligonucleotide amplification methods that can be used in the context of the present invention are well-known to the skilled person and exemplified herein (see the items and the appended examples for a non-limiting illustration of these methods).
- these methods are characterized by a method for detecting the methylation status of at least one defined 5 ' -CpG-3 ' (comprised by a target sequence which target sequence is comprised by a vertebrate genomic nucleic acid) comprising the steps of:
- the present inventors discovered that the methylation or hydroxymethylation of the "C" of said at least one defined 5 ' -CpG-3 ' influences the extension reaction from the 3 ' -end when using the primer of the present invention.
- the rationale is as follows, without being bound by theory.
- the primer of the present invention has its 3 " end opposite to the C of said 5 ' -CpG-3 ' , and comprises at said 3 " end a mismatched nucleotide in relation to said C of said 5 ' -CpG-3 ' .
- Said mismatched nucleotide at the utmost 3'-end of the primer does not canonically pair with the cytosine of interest, in case said cytosine is not methylated or hydroxymethylated, which negatively impairs the 3 ' -extension reaction of said primer.
- the methylation/hydroxymethylation of said "C” somehow compensates for said mismatch which positively impairs the 3 ' -extension reaction of said primer (see for example Fig. 2 for further illustration).
- the methylation/hydroxymethylation status of the "C" in the at least one 5 ' -CpG-3 ' thus influences the extension reaction from the 3 " -end of the primer of the invention.
- Said influence can be detected by standard methods (some of them are exemplified herein), e.g. by evaluating the amount of amplified product obtained in the respective methods and/or by evaluating the velocity of reaction (e.g. the velocity of dNTP incorporation) and/or by evaluating the process capability index etc. All these methods are well-known and established.
- the amplified product is detected with the vertebrate genomic nucleic acid as a template and also with at least one further template (control template) that comprises at least the target sequence comprising said at least one defined 5 ' -CpG-3 ' and a downstream template sequence that can be amplified (said template sequence can be identical to the template sequence of the vertebrate genomic nucleic acid sequence or not).
- control template comprises at least the target sequence comprising said at least one defined 5 ' -CpG-3 ' and a downstream template sequence that can be amplified (said template sequence can be identical to the template sequence of the vertebrate genomic nucleic acid sequence or not).
- the "C" of said at least one defined 5 ' -CpG-3 ' of the control template is either methylated/hydroxymethylated or not.
- methylated/hydroxymethylated it may serve as a sort of "positive control” (displaying the positive effect of a methyl residue on the 3 ' - extension when using the 3 ' -mismatched primer of the present invention). If it is not methylated/hydroxymethylated, then it may serve as a sort of negative control (displaying the negative effect of a non-methylated/non-hydroxymethylated residue on 3 ' -extension when using the 3 ' -mismatched primer of the present invention).
- control template (A) comprising 5% methylated "C”
- control template (B) comprising 10% methylated "C”
- control template (C) comprising 20% methylated "C” etc.
- control templates display the influence of the quantitative methylation status at the very "C” on the 3 ' -end extension reaction obtained with the primer of the invention.
- these values obtained with these controls for example the calibration curve obtained therewith
- the respective value obtained with the vertebrate genomic nucleic acid as a template then it will be possible to quantify the methylation status of said "C” comprised in the 5 ' -CpG-3 ' of interest in said vertebrate genomic nucleic acid.
- the methods and uses and kits of the present invention may thus also be used for the quantification (or the semi-quantification) of the methylation status of a defined "C" comprised in the 5 ' -CpG-3 ' of interest in said vertebrate genomic nucleic acid.
- Detection of the amplified products may be accomplished by using any known method.
- the amplified nucleic acids may be associated with a surface that results in a detectable physical change, e.g., an electrical change.
- Amplified nucleic acids may be detected in solution phase or by concentrating them in or on a matrix (such as a gel, e.g. a polyacrylamide gel - see Fig. 3C) and detecting labels associated with them (e.g., an intercalating agent such as ethidium bromide or SYBR green or labeled primer).
- a matrix such as a gel, e.g. a polyacrylamide gel - see Fig. 3C
- labels associated with them e.g., an intercalating agent such as ethidium bromide or SYBR green or labeled primer.
- Other detection methods use probes complementary to a sequence in the amplified product and detect the presence of the probe:product complex, or use a complex of probes to amplify the signal detected from amplified products (e.g., U.S. Pat. No. 5,424,413).
- Other detection methods use a probe in which signal production is linked to the presence of the target sequence because a change in signal results only when the labeled probe binds to amplified product, such as in a molecular beacon, molecular torch, hydrolyzation probes (Taqman) or hybridization switch probe (e.g., U.S. Pat. Nos. 5,1 18,801 ).
- Such probes typically use a label (e.g., fluorophore) attached to one end of the probe and an interacting compound (e.g., quencher) attached to another location of the probe to inhibit signal production from the label when the probe is in one conformation ("closed") that indicates it is not hybridized to amplified product, but a detectable signal is produced when the probe is hybridized to the amplified product which changes its conformation (to "open”). Detection of a signal from directly or indirectly labeled probes that specifically associate with the amplified product indicates the presence of the target nucleic acid that was amplified.
- a label e.g., fluorophore
- quencher interacting compound
- the DNA polymerase that may be used in the context of the present invention is not particularly limited. Respective DNA polymerases are known in the art (including the respective wt Polymerases and variations thereof comprising specific mutations as described and known in the art).
- the DNA polymerase is a replicative DNA polymerase, more preferably selected from the group consisting of family A DNA polymerases and family B DNA polymerases, more preferably selected from the group consisting of Taq DNA polymerase; KlenTaq DNA polymerase, Thermococcus kodakaraensis (KOD) DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Tth DNA polymerase etc. to name some.
- KOD Thermococcus kodakaraensis
- said DNA polymerase is a protein which is at least 85%, 95%, 96%, 97%, 98%, 99% or even 100% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ ID No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively.
- SEQ ID No. 1 depicts the sequence of the KOD wt polymerase.
- a DNA polymerase which is 100% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ ID No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively, was used in a method for detecting the methylation status of a given template.
- Said Polymerase is denoted herein KOD exo- C6 (or KOD exo- symptom C6). It turned out that this polymerase scores very well in the methods of the present invention (see Figure 6).
- the present invention thus also relates to a protein which is at least 85%, 95%, 96%, 97%, 98%, 99% or even 100% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ I D No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively.
- the present invention also relates to nucleic acids encoding the protein which is at least 85% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ ID No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively.
- Vectors comprising said nucleic acid are also contemplated.
- Host cells comprising these vectors are also included herein.
- the present invention relates to the use of a primer as defined herein and the polymerase as defined above in an oligonucleotide amplification method.
- said amplification method is for the specific amplification of a vertebrate genomic nucleic acid which comprises a target sequence which target sequence comprises at least one defined 5 ' -CpG-3 ' .
- kits typically defines a package including one or more of the primer of the invention, and/or other compositions associated with the invention, for example, a nucleic acid probe, and/or a positive control, and/or a negative control, and/or a polymerase, and/or a microarray, etc, as described herein.
- the kit may be directed to: determining the methylation status of at least one 5 ' -CpG-3 ' ; determining the methylation status of one or more selected nucleic acids molecules (for example, of genomic DNA, mitochondrial DNA, etc.
- the kit further contains reagents for isolating the vertebrate genomic nucleic acid.
- Each of the ingredients of the kit may be provided in liquid form (e.g., in solution), or in solid form (e.g., a dried powder).
- some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species, which may or may not be provided with the kit.
- compositions or components associated with the invention include, but are not limited to, solvents, surfactants, diluents, salts, buffers, emulsifiers, chelating agents, fillers, antioxidants, binding agents, bulking agents, preservatives, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, dishes, frits, filters, rings, clamps, wraps, patches, containers, and the like, for example, for using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the compositions components for a particular use.
- a kit of the invention may, in some cases, include instructions and/or an imprint in any form.
- the instructions may include instructions for the use, modification, mixing, diluting, preserving, assembly, storage, packaging, and/or preparation of the compositions and/or other compositions associated with the kit.
- the instructions may also include instructions, for example, for a particular use disclosed herein. The same applies to the imprint.
- the instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions, for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications), provided in any manner.
- the present invention thus relates to a kit comprising at least one primer as defined herein and optionally means to conduct the amplification.
- said means comprises at least the DNA polymerase as defined herein, in particular the DNA polymerase which is a protein which is at least 85%, 95%, 96%, 97%, 98%, 99% or even 100% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ ID No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively.
- the present invention relates to a kit comprising a primer as defined herein and an isolated oligonucleotide as a positive control and/or a negative control, wherein said positive control comprises the target sequence for said at least one primer including said at least one defined 5 ' -CpG-3 ' and wherein the C in said at least one defined 5 ' - CpG-3 ' is methylated or hydroxymethylated, and wherein said negative control comprises the target sequence for said at least one primer including said at least one defined 5 ' -CpG-3 ' and wherein the C in said at least one defined 5 ' -CpG-3 ' is neither methylated nor hydroxymethylated.
- DNA methylation and chromatin structure are often altered in diseases, particularly in cancer.
- Cancer in general, is caused by dysfunction of genes which control the cell cycle, apoptosis and migration.
- oncogenes are activated and enhance division or prevent cell death.
- Tumor suppressor genes can be inactivated and are no longer available to stop these procedures.
- cytosine methylation can contribute to the development of cancer.
- the genome can be hypomethylated and this leads to genomic instability, or the promoters of tumor suppressor genes become hypermethylated which leads to silencing of these genes.
- methylated CpG sites are mutation hot spots, as spontaneous deamination of 5- methylcytosine to the natural base thymine is not recognized.
- methylated 5 ' -CpG-3 ' sites increase the rate of UV-induced mutations and the binding of some chemical carcinogens.
- Epigenetic silencing and genetic mutations are often recessive and require the disruption of both alleles for full expression of the changed phenotype.
- Three classes of hits participate in different combinations to inhibit completely the function of tumor suppressor genes.
- the first hit of inactivation can be a direct mutation or gene silencing by DNA methylation.
- the second step could be the loss of heterozygosity or DNA methylation again.
- DNA is more stable than RNA or protein, and methyl groups on cytosines are part of the covalent DNA which is not the case for chromatin. Furthermore, DNA methylation analysis is independent of the total amount of starting material because the ratio of methylated and unmethylated CpG sites is determined. 5- Methylcytosine represents a positive epigenetic marker that can be detected independently of expression levels and more easily than a negative signal like loss of heterozygosity. Another advantage is the theoretical reversal of epigenetic changes by treatment with pharmaceuticals, whereas genetic changes are irreversible.
- the methylations status of said at least one defined 5 ' -CpG-3 ' is associated with a disease (or disease state) and/or is comprised in a genetic element which is associated with a disease (or disease state), such as for example a tumor suppressor gene, a genetic element controlling the expression of a tumor associated antigen etc..
- the present invention relates to the use of at least one primer of the invention for in vitro diagnosis of a disease of a vertebrate, which disease is associated with the methylation status of the genomic nucleic acid at said C.
- the diseases mentioned include but are not limited to neurological diseases, metabolic diseases, cardiovascular diseases, autoimmune diseases, cancer etc.
- the term "cancer" in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may circulate in the blood stream as independent cells, such as leukemic cells.
- the methods, uses and kits of the present invention may also be used for identifying nucleic acid molecules differentially methylated in a disease.
- the methods/uses and kits of the present invention may for example be utilized to identify candidate tumor suppressor genes.
- the methods, uses and kits of the invention are particularly valuable for identifying new markers whose methylation status is linked to disease.
- the embodiments of the present invention may also be used for classifying epithelial and mesenchymal phenotypes in cancer (see e.g. WO2013055530 for illustration); for predicting the sensitivity of tumor cell growth to inhibition by inhibitors (see e.g. WO2013055530 for illustration).
- the uses, kits and methods of the present invention may thus be used for diagnosis of diseases. Since specific alterations in the methylation status of the respective "C" comprised by at least one defined 5 ' -CpG-3 ' may be associated with disease state, these methods, kits and uses serve as reliable platform for diagnosis, prognosis and the analysis associated with diseases, which diseases are characterized by an altered or predictive methylation status.
- diagnosis is used herein to refer to the identification of a molecular or pathological state, disease or condition.
- prognosis is used herein to refer to the prediction of the likelihood of disease-attributable symptoms.
- the methods, uses and kits provided can also be used to profile populations to aid the development and application of patient-oriented treatments.
- IGFBP3 Colon, lung, ovarian, prostate
- N0RE1A Colon, liver, lung, thyroid
- PYCARD Glioma breast, colon, gastric, lung
- RASSF1A Breast, ovarian, lung, prostate, colon
- RIZ1 Leukaemia, liver, thyroid, gastric, prostate
- FMR1 prediction of treatment of Fragile X Syndrome FXS
- methylation status may also be used to evaluate the age of a subject (WO 2012162139) or to discriminate fetal from maternal DNA (US 201 10143342). Further applications of the methods and uses and kits of the present invention are disclosed in WO2001068912. It will be understood however, that the uses, kit and methods of the present invention may be used in all thinkable situations where it is already known or assumed that the methylation status of a defined "C" is of interest, for example because it is indicative of a disease.
- the present invention aims at determining or diagnosing the methylation status of at least one of the genes selected from BRCA1 , MGMT PYCARD, RASSF1A, SHOX2 and/or SEPT9.
- the present invention further relates to the use of at least one primer as defined herein, for the stratification of patients suffering from a disease, which disease is associated with the methylation status of the genomic nucleic acid at said C. It is envisaged that the above embodiment makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template together with said primer. [0051] The present invention also relates to the use of at least one primer as defined herein, for predicting the therapeutic response of patients suffering from a disease, which disease is associated with the methylation status of the genomic nucleic acid at said C. It is envisaged that the above embodiment makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template together with said primer.
- the present invention relates to a primer as defined herein for use in a method of the invention and in particular for determining the methylation status of a vertebrate genomic nucleic acid at said C and/or for (in vitro) diagnosis of a disease of a vertebrate, which disease is associated with the methylation status of the genomic nucleic acid at said C. It is envisaged that the above embodiment makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template together with said primer.
- the present invention also relates to the following items and the corresponding embodiments.
- Item 1 A method for directly detecting methylation or hydroxymethylation of a cytosine residue of interest in a DNA molecule, comprising the steps of:
- Item 2 The method of item 1 , wherein the mismatched base is selected from the group consisting of adenine, cytosine, thymine, uracil and modifications thereof.
- Item 3 The method of item 1 or claim 2, wherein said specific DNA polymerase reaction is selected from the group consisting of primer extension, rolling circle extension (RCA) and polymerase chain reaction (PCR).
- said specific DNA polymerase reaction is selected from the group consisting of primer extension, rolling circle extension (RCA) and polymerase chain reaction (PCR).
- Item 4 The method of item 3, wherein said PCR is quantitative real-time PCR (qRT-PCR).
- Item 5 The method of any one of items 1 to 4, wherein the DNA polymerase used for said specific DNA polymerase reaction is selected from the group consisting of family A DNA polymerases and family B DNA polymerases.
- Item 6 The method of item 5, wherein the DNA polymerase is selected from the group consisting of KlenTaq DNA polymerase, KOD DNA polymerase, Vent DNA polymerase, and Deep Vent DNA polymerase.
- the DNA polymerase is selected from the group consisting of KlenTaq DNA polymerase, KOD DNA polymerase, Vent DNA polymerase, and Deep Vent DNA polymerase.
- Item 7 The method of any one of items 1 to 6, wherein the increased efficiency of the specific DNA polymerase reaction indicating methylation or hydroxymethylation of the DNA molecule is an increased efficiency by 1 to 30 cycles, preferably 5 to 20 cycles, more preferably 10 to 20 cycles.
- the present invention thus also relates to a method for directly detecting methylation or hydroxymethylation of a cytosine residue of interest in a DNA molecule, comprising the steps of:
- the terms “directly detecting” or “direct detection” relate to the fact that with the method of the present invention as described in the items, methylation or hydroxymethylation of a cytosine residue of interest in a DNA molecule can be directed without the need for any pretreatment or chemical modification of the DNA molecule. Accordingly, the method of the present invention as described in the items is significantly less time-, labor- and cost-intensive compared to methods known in the art. Moreover, the method of the present invention as described in the items is much less prone to errors and allows the analysis of very small amounts of sample material.
- the primer provided in step (a) of the method of the present invention as described in the items is specifically designed for the analysis of a particular cytosine residue of interest in a known DNA molecule.
- said primer binds to the DNA molecule in a manner that its 3'-end is opposite of the cytosine residue and said 3'-end has a mismatched base in respect to the cytosine of interest.
- Said mismatched base at the 3'-end of the primer does not canonically pair with the cytosine of interest, in case said cytosine is not methylated or hydroxymethylated, thus impairing the specific DNA polymerase reaction with said primer using said DNA molecule as template.
- mismatched base at the 3'-end of the primer is thought to pair in a non-canonical manner with the cytosine of interest, in case said cytosine is methylated or hydroxymethylated, thus allowing a more efficient specific DNA polymerase reaction with said primer using said DNA molecule as template (Fig. 2).
- the mismatched based is selected from the group consisting of adenine, cytosine and thymine, and modifications thereof.
- primers can be labeled with a detectable marker as known in the art, e.g. with a radioactive or dye label.
- the primer is selected from the group of primers as shown in SEQ ID NOs. 2 to 5 (SEQ ID NO. 2: TTG CTC CCG TCG GCG CTT CTT TCA; SEQ ID NO. 3: GTT TCT CCA GTT TCT TTT CTC A; SEQ ID NO. 4: GTT TCT CCA GTT TCT TTT CTC C; SEQ ID NO. 5: GTT TCT CCA GTT TCT TTT CTC T).
- the mismatched base is an artificial nucleobase that has the characteristic of mismatching with methylated or hydroxymethylated cytosine.
- Respective artificial nucleobases are not particularly limited and are known in the art.
- the specific DNA polymerase reaction performed in step (b) and/or (c) of the method of the present invention as described in the items is not particularly limited, provided that it allows the discrimination between unmodified and methylated or hydroxymethylated cytosine residues.
- Suitable DNA polymerase reactions include established standard methods and are known in the art.
- the specific DNA polymerase reaction is selected from the group consisting of primer extension, rolling circle amplification (RCA) and PCR-based methods such as quantitative real-time PCR (qRT-PCR).
- RCA rolling circle amplification
- qRT-PCR quantitative real-time PCR
- the specific DNA polymerase reaction is a primer extension reaction
- said primer extension reaction is preferably performed for 10 to 90 seconds.
- a suitable additional primer i.e.
- the DNA polymerase used for the specific DNA polymerase reaction is not particularly limited. Respective DNA polymerases are known in the art.
- the DNA polymerase is a replicative DNA polymerase, more preferably selected from the group consisting of family A DNA polymerases and family B DNA polymerases, more preferably selected from the group consisting of KlenTaq DNA polymerase, Thermococcus kodakaraensis (KOD) DNA polymerase, Vent DNA polymerase, and Deep Vent DNA polymerase.
- KOD Thermococcus kodakaraensis
- step (c) of the method of the present invention as described in the items, methylation or hydroxymethylation of the cytosine residue of interest is indicated by an increased efficiency of said specific DNA polymerase reaction compared to a corresponding DNA polymerase reaction performed with said primer using a corresponding DNA molecule, wherein the cytosine residue of interest is not methylated or hydroxymethylated, as template.
- the efficiency of the specific DNA polymerase reaction is assessed in comparison to a corresponding unmodified DNA molecule, i.e. a DNA molecule wherein the cytosine residue of interest is neither methylated nor hydroxymethylated.
- corresponding DNA molecule as used in this context relates to a DNA molecule having the same sequence as the DNA molecule to be analyzed at least in the region of primer binding and the upstream region that is replicated in the DNA polymerase reaction.
- the increased efficiency of the specific DNA polymerase reaction indicating methylation or hydroxymethylation of the DNA molecule is an increased efficiency by 1 to 30 cycles, preferably 5 to 20 cycles, more preferably 5 to 15 cycles.
- the increased efficiency of the specific DNA polymerase reaction indicating methylation or hydroxymethylation of the DNA molecule is an increased efficiency by 1 to 30, 5 to 30, 5 to 25, 10 to 25, 10 to 20, or 15 to 20 cycles.
- methylation or hydroxymethylation of the cytosine residue of interest is indicated by an increased efficiency of primer extension reactions which can be quantified in an absolute manner.
- Methods of the present invention as described in the items provide a means for the direct detection of methylated or hydroxymethylated cytosine residues of interest in a DNA molecule in a fast, simple, and accurate manner. Said methods can be conducted without a bisulphite pretreatment of the DNA templates, i.e. these methods do not need a pretreatment, such as a bisulphite pretreatment or chemical modification of the DNA molecule to be analyzed. Therefore, it is less prone to errors as compared to methods known in the art, and allows the analysis of smallest amounts of sample material. Moreover, said method can be performed using well established standard methods for the DNA polymerase reaction.
- the C * indicates the cytosine of interest and is either methylated or unmethylated.
- the primer ends with an adenine to generate a mismatch opposite of the cytosine of interest in the template.
- a 24 nt radioactive labeled primer was used.
- Full-length product is at 31 nt.
- the 32 nt product is formed by a non- templated nucleotide addition to the 3'-termini of the blunt-ended DNA strands and has been observed before for KlenTaq DNA polymerase.
- Reaction products are separated by denaturing PAGE. Reactions for methylated and unmethylated template were started in parallel and stopped after certain time periods. Clearly more product is formed with the methylated template compared to the unmethylated. D) Quantification of extended primer. The ratio of extended to unextended primer was determined with Quantity One software.
- Figure 4 Direct detection of methylcytosine by quantitative real-time PCR
- the C * in the template indicates the cytosine of interest and is either methylated, hydroxymethylated or unmethylated.
- the N in the forward primer stands for adenine, cytosine, guanine, thymine, or modifications thereof. In the case of adenine, cytosine, thymine, or modifications thereof the primer is mismatched at the 3'-end. For guanine the complete primer is matched.
- the used primer is named in the headline of each graph. No discrimination is detected for the matched primer G (upper left corner). For the three mismatch primers clear discrimination between methylated and unmethylated cytosine in the template is visible.
- Oligonucleotides were purchased from Thermo Fisher Scientific or Metabion, Germany. dNTPs were either from Roche (primer extensions) or Fermentas (quantitative real-time PCR). The KlenTaq DNA polymerase was overexpressed in E. coli and purified with Ni-IDA as known in the art. Enzyme purity and quantity were determined by SDS-PAGE using an albumin standard dilution curve. Quantitative real-time PCR was performed on a Chromo4 instrument from Bio- Rad. SYBRgreen I was purchased from Fluka. Denaturing PAGE was analyzed with a Molecular Imager Fx from Bio-Rad.
- Reaction mixtures (20 ⁇ _) contained 50 mM Tris-HCI (pH 9.2), 16 mM (NH 4 ) 2 S0 4 , 0.1 % Tween20, 2.5 mM MgCI 2 , 400 nM KlenTaq DNA polymerase, 150 nM primer (24 nt, 5'- [ 32 P]d(TTG CTC CCG TCG GCG CTT CTT TCA)-3'], SEQ ID NO: 2, and 200 nM template (34 nt, 5'-d(GGC AAC GAG GGC AGC CGC GAA GAA AG Me C ATC CGG C)-3') (Fig. 3 A).
- Results can be taken from Fig. 3 C, showing that clearly more product is formed with the methylated template compared to the unmethylated template.
- Quantitative real-time PCR with methylated and unmethylated DNA template Reaction mixtures (20 ⁇ _) contained 50 mM Tris-HCI (pH 9.2), 16 mM (NH 4 ) 2 S04, 0.1 % Tween20, 2.5 mM MgCI 2 , 250 ⁇ of each dNTP, 0.6x SYBRgreen I and 200 nM KlenTaq DNA polymerase.
- RT-Epi90C As templates, either RT-Epi90C [60 pM, 90 nt, 5'-d(GGG GCA GAG CGA GCT CCC GAG TGG GTC TGG AGC CGC GGA GCT GGG CGG GGG CGG GAA GGA GGT AGC GAG AAA AGA AAC TGG AGA AAC TCG)-3'] or RT Epi90MeC [60 pM, 90 nt, 5'-d(GGG GCA GAG CGA GCT CCC GAG TGG GTC TGG AGC CGC GGA GCT GGG CGG GGG CGG GAA GGA GGT AG Me C GAG AAA AGA AAC TGG AGA AAC TCG) were used (Fig. 4 A). Both templates had the same sequence except of the methylation pattern at the indicated position.
- RT-Epi22Prev [5'- d(GCA GAG CGA GCT CCC GAG TG)-3'] was used together with one of the following forward Primers RT-Epi22Afor [5'-d(GTT TCT CCA GTT TCT TTT CTC A)-3'; SEQ ID NO: 3], RT- Epi22Cfor [5'-d(GTT TCT CCA GTT TCT TTT CTC C)-3'; SEQ ID NO: 4], RT-Epi22Gfor [5'- d(GTT TCT CCA GTT TCT TTT CTC G)-3'] or RT-Epi22Tfor [5'-d(GTT TCT CCA GTT TCT TTT CTC T)-3'; SEQ ID NO: 5] (Fig. 4 A). All forward primers differed only at the last nucleotide at the 3' end.
- the product was amplified by 50 PCR cycles (95°C for 15 s, 55°C for 10 s and 72°C for 15 s), and analyzed by melting curve measurement from 55° to 95°C with a read every 0.5°C.
- Results can be taken from Fig. 4 B, showing that no discrimination between methylated and unmethylated template is seen for the matched primer (having a G at the 3'-end), whereas a clear discrimination can be seen for the mismatched primers.
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Abstract
La présente invention concerne des moyens et des procédés pour la détection de l'état de méthylation de résidus cytosine et notamment l'utilisation d'au moins une amorce qui est d'une longueur d'au moins 5 nucléotides, et qui s'hybride avec une séquence cible qui comprend au moins un 5'-CpG-3' défini, ladite séquence cible étant comprise dans un ADN génomique d'un vertébré, et dont l'extrémité 3' est opposée au C dudit 5'-CpG-3', et qui comprend au niveau de ladite extrémité 3' un nucléotide non correspondant par rapport audit C dudit 5'-CpG- 3', pour la détermination de l'état de méthylation d'un acide nucléique génomique d'un vertébré au niveau dudit C. La base non correspondante située à l'extrémité 3' de l'amorce ne s'apparie pas à ladite cytosine dans le cas où la cytosine n'est pas méthylée ou hydroxyméthylée, ce qui empêche une réaction impliquant l'ADN polymérase avec ladite amorce, à l'aide de ladite molécule d'ADN qui sert de modèle. Cependant, la base non correspondante située à l'extrémité 3' de l'amorce s'apparie à ladite cytosine dans le cas où la cytosine est méthylée ou hydroxyméthylée, ce qui permet une réaction impliquant l'ADN polymérase avec ladite amorce, à l'aide de ladite molécule d'ADN qui sert de modèle.La présente invention concerne également des protéines polymérases qui peuvent être utilisées dans les modes de réalisation de la présente invention. La présente invention concerne également un kit comprenant au moins une amorce de l'invention et éventuellement des moyens de réalisation de l'amplification.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/EP2012/003279 WO2014019595A1 (fr) | 2012-08-01 | 2012-08-01 | Procédé de détection de méthylation et d'hydroxyméthylation de l'adn |
EPPCT/EP2012/003279 | 2012-08-01 |
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WO2014183093A1 (fr) * | 2013-05-10 | 2014-11-13 | University Of Southern California | Biomarqueurs de méthylation de l'adn pour le cancer de la vessie |
CN109554473A (zh) * | 2018-12-18 | 2019-04-02 | 敬善生物科技江苏有限公司 | 一种在宫颈癌检测中应用的试剂盒及其应用 |
CN109609638A (zh) * | 2019-01-05 | 2019-04-12 | 敬善生物科技江苏有限公司 | 一种在鼻咽癌检测中应用的试剂盒及其应用 |
CN110964826A (zh) * | 2019-12-27 | 2020-04-07 | 大连晶泰生物技术有限公司 | 一种结直肠癌抑癌基因甲基化高通量检测试剂盒及其应用 |
CN110982883A (zh) * | 2019-12-30 | 2020-04-10 | 西安交通大学 | 一种高通量单细胞基因组5-羟甲基嘧啶单分子可视化分析方法 |
US20210032704A1 (en) * | 2018-01-23 | 2021-02-04 | Exellen Medical | Method and kit for identifying lung cancer status |
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WO2014183093A1 (fr) * | 2013-05-10 | 2014-11-13 | University Of Southern California | Biomarqueurs de méthylation de l'adn pour le cancer de la vessie |
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CN110964826B (zh) * | 2019-12-27 | 2023-07-25 | 大连晶泰生物技术有限公司 | 一种结直肠癌抑癌基因甲基化高通量检测试剂盒及其应用 |
CN110982883A (zh) * | 2019-12-30 | 2020-04-10 | 西安交通大学 | 一种高通量单细胞基因组5-羟甲基嘧啶单分子可视化分析方法 |
EP3971302A1 (fr) * | 2020-09-16 | 2022-03-23 | QIAGEN GmbH | Procédé de détection de cytosine 5-hydroxyméthylée |
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