Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-28
2001-10-09
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024200, C536S024330
Reexamination Certificate
active
06300071
ABSTRACT:
The present invention relates to a method for detecting DNA methylation using AFLP™. In particular, this method can be used to distinguish between methylated and non-methylated sites (nucleotides) in a nucleic acid, more particular between methylated and non-methylated restriction sites. Thus, the method of the invention can provide information on the methylation pattern of the DNA, which can be visualised as a DNA-fingerprint.
Selective restriction fragment amplification or AFLP is known, for instance from the European patent application 0 534 858 by applicant, incorporated herein by reference. In general, AFLP comprises the steps of:
(a) digesting a nucleic acid, in particular a DNA, with one or more specific restriction endonucleases, to fragment said DNA into a corresponding series of restriction fragments;
(b) ligating the restriction fragments thus obtained with at least one double-stranded synthetic ligonucleotide adapter, one end of which is compatible with one or both of the ends of the restriction fragments, to thereby produce tagged restriction fragments of the starting DNA;
(c) contacting said tagged restriction fragments under hybridizing conditions with at least one oligonucleotide primer;
(d) amplifying said tagged restriction fragments hybridized with said primers by PCR or similar technique so as to cause further elongation of the hybridized primers along the restriction fragments of the starting DNA to which said primers hybridized; and
(e) identifying or recovering the amplified or elongated DNA fragment thus obtained.
The amplified DNA-fragments thus obtained can then be analysed and/or visualised, for instance by means of gel-electrophoresis. This provides a genetic fingerprint showing specific bands corresponding to the restriction fragments which have been linked to the adapter, have been recognized by the primer, and thus have been amplified during the amplification step. The fingerprint thus obtained provides information on the specific restriction site pattern of the starting DNA, and thus on the genetic make-up of the organism from which said DNA has been derived.
AFLP can therefore be used to identify said DNA; to analyse it for the the presence of specific restriction site patterns, restriction fragment length polymorfisms (RFLP's) and/or specific genetic markers (so-called “AFLP-markers), which may be indicative of the presence of certain genes or genetic traits; or for similar purposes, for instance by comparing the results obtained to DNA-samples of known origin or restriction pattern, or data thereon.
The primers used in AFLP are such that they recognize the adapter and can serve as a starting point for the polymerase chain reaction. To this end, the primers must have a nucleotide sequence that can hybridize with (at least part of) the nucleotide sequence of the adapter adjacent to the 3′ end of the restriction fragment to be amplified. The primers can also contain one or more further bases (called “selective bases”) at the 3′-end of their sequence, for hybridization with any complementary base of bases at the 3′-end of the adapter ligated restriction fragment. As, of all the adapter ligated restriction fragments present in the mixture, only those fragments that contain bases complementary to the selective bases will subsequently be amplified, the use of these “selective” primers will reduce the total amount of bands in the final fingerprint, thus making the fingerprint more clear and more specific. Also, the use of different selective primers will generally provide differing fingerprints, which can also be used as a tool for the purposes of identification or analysis.
As AFLP provides amplification of both strands of a double stranded starting DNA, AFLP advantageously allows for exponential amplification of the fragment, i.e. according to the series 2, 4, 8, 16, etc.. Also, AFLP requires no prior knowledge of the DNA sequence to be analysed, nor prior identification of suitable probes and/or the construction of a gene library from the starting DNA.
For a further description of AFLP, its advantages, its embodiments, as well as the techniques, enzymes, adapters, primers and further compounds and tools used therein, reference is made to EP-0 534 858, incorporated herein by reference. Also, in the description hereinbelow, the definitions given in paragraph 5.1 of EP-0 534 858 will be used, unless indicated otherwise.
It is well known that the DNA of a prokaryotic or eukaryotic organism can contain methylated sites, i.e. that certain nucleotides of said DNA strands can be substituted with a methyl-group. In particular, cytosine residues, as well as adenine residues (in bacteria), can be methylated; for instance, in mammals, it is known that 2-7% of all cytosine-residues are methylated, and this may be as high as 30% in plants. Methylated cytosines can occur as mCG doublets, as small palindromic
5′-
m
CpG -3′
3′-GpC
m
-5′ sequences, with both cytosine residues being methylated, or as
m
CNG triplets (the latter particular in plants). Often, the majority of CG-sites in the DNA of both eukaryotes and bacteria are methylated.
In prokaryotic organisms, the pattern of DNA-methylation can be used to identify a particular bacterial strain or to distinguish replicated and non-replicated DNA (vide B. Lewin, GENES V, Oxford Univ. Press 1944, chapter 20). DNA-methylation also plays a role in DNA repair and the timing of DNA replication.
In eukaryotes, DNA-methylation is known to be involved in several genetic mechanisms, such as the regulation of gene expression, for instance through gene silencing or gene activation (vide B. Lewin, GENES V, Oxford Univ. Press 1994, chapter 28).
Also, in eukaryotes, DNA methylation is thought to be associated with genetic diseases through the mechanism of “imprinting”, as well as increased susceptibility for mutagenesis and the origin of cancer. For instance, in female individuals, DNA-methylation, which is involved in X-chromosome activation/inactivation, can be used for distinguishing between neoplastic (clonal) cell populations and pseudoplastic or hyperplastic populations, to determine whether a growth of these cells is malignant or not (WO 96/27024). Also, the state of methylation of reporter genes has been used in in vivo mutagenicity assays (WO 93/17123 and the references cited therein).
Furthermore, changes in DNA-methylation can occur during gene transformation, making it possible to distinguish transformed and not-transformed genes or sequences. For instance, analysis of (the changes in) DNA methylation pattern has been used for the early detection of transgenic embryo's (WO 92/22647).
In plants, such as pea and tomato, methylation patterns can be used to distinguish between varieties by detecting restriction fragment length polymorphisms characteristic of these distinctive varieties (WO 90/05195). In tomato, this is carried out by digesting genomic DNA with a non-methylation sensitive restriction enzyme, and screening the fragments thus obtained by means of Southern hybridization using detectably labelled probes, said probes having been obtained by digestion of tomato genomic DNA with a methylation sensitive restriction enzyme. The bands in the resulting DNA-fingerprint enable identification of species specific, variety specific or individual RFLP's.
However, this method is not suited or intended for specifically distinguishing between methylated and non-methylated sites within the target DNA. Also, the genomic DNA to be analysed is itself not treated with a methylation sensitive restriction enzyme (these are only used in generating the probes). Furthermore, the technique described in WO 90/05195 does not involve any DNA-amplification step, and suffers from the general disadvantages of similar conventional RFLP detection techniques, such as low resolution, as well as being time consuming and laborious (compare EP-A-0 534 858, paragraph 2.1).
Nucleotide methylation using sequence specific methylases or restriction methylases has also been used as a tool for ma
Vos Petrus Antonius Josephina
Vuylsteke Marnik Johan Roger
Zabeau Marcus Florent Oscar
Browdy and Neimark
Forman B J
Jones W. Gary
Keygene N.V.
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