Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-09
2002-02-05
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S094000
Reexamination Certificate
active
06344319
ABSTRACT:
The present invention relates in particular to a method for detecting and locating polynucleotide sequences (which may contain or otherwise genes or gene portions) in a genome or a genome portion using the so-called molecular combing technique.
The present invention also relates to a method for detecting and locating reagents of biological, natural or synthetic origin by combining said reagents with all or part of the combed DNA.
The technique of molecular combing, as described in the following references: PCT/FR95/00164 of Oct. 2, 1995 and PCT/FR95/00165 of Oct. 2. 1995, applied to nucleic acids, and more particularly to genomic DNA, allows the uniform extension and the visualization of DNA or of RNA in the form of rectilinear and practically aligned filaments.
The present invention is based on the demonstration of the fact that, using probes, that is to say polynucleotides containing a chain of nucleotide sequences such as labeled DNA molecules which specifically recognize portions of the aligned DNA, which are hybridized with the combed DNA, it is possible to directly visualize, on the combed genome, the position of the complementary sequence.
Under these conditions, it is possible, for example using two probes labeled with different chromophores such that they can be visualized by a color, red and green for example, to measure the distance separating them. However, it is also possible, using different probes or a series of contiguous probes (called hereinafter “contig”), to directly measure the length of the region of interest, and to measure the potential impairments thereof in the case of an abnormal genome.
The present invention therefore relates, in particular, to the diagnosis of genetic diseases which are preferably characterized by substantial impairments of the genome, either in its structure, deletion or translocation for example, or in the number of copies of certain sequences (trisomy for example, where the sequence represents the whole of a chromosome), as well as to methods which allow genes to be located and mapped rapidly.
Genetic diagnosis may be divided into several fields:
prenatal,
pathologies with a genetic component,
cancer and susceptibility to cancer.
Prenatal Diagnosis
The majority (95%) of fetal abnormalities are due to trisomies of chromosomes 21, 18, 13, X or Y. Their conclusive diagnosis is somewhat late (17th week of amenorrhea, by amniocentesis for example). Aminiocentesis requires a substantial puncture of amniotic fluid (a few tenths of milliliters) from which fetal cells in suspension are extracted and cultured for several days (see the technique described by S. Mercier and J. L. Bresson (1995) Ann. Génét., 38, 151-157). A karyotype of these cells is established by macroscopic observation and counting the chromosomes by a highly specialized staff.
A technique involving the collection of chorial villi makes it possible to dispense with the culturing step and avoids the collection of amniotic fluid. Karyotype analysis requires, however, the same work (see Médecine Prénatale. Biologie Clinique du Foetus. André Boué, Publisher Flammarion, 1989). These two techniques may be applied earlier (up to 7 weeks of gestation for the collection of chorial villi and 13-14 weeks for aminiocentesis), but with a slightly increased risk of abortion. Finally, a direct collection of fetal blood at the level of the umbilical cord allows karyotyping without culturing, but presupposes a team of clinicians specialized in this technique (C. Donner et al., 1996, Fetal Diagn. Ther., 10, 192-199).
Other abnormalities such as translocations or deletions/insertions of substantial portions of chromosomes may be detected at this stage, or by using techniques such as fluorescent in situ hybridization (FISH). However, here again, this type of diagnosis can only be carried out by a highly qualified staff.
Studies show, moreover, that there are as yet no immunological methods allowing the detection of fetal markers in maternal blood allowing a conclusive diagnosis of trisomy 21 or of other abnormalities (see, for example, N. J. Wald et al., 1996, Br. J. Obstet. Gynaecol., 103, 407-412 for trisomy 21—related Down's syndrome).
The current prenatal diagnoses therefore have numerous disadvantages: they can only be carried out at a relatively late stage of the development of the embryo; they are not completely without risk for the fetus or for the mother; the results are often obtained after a fairly long time (about 1 to 3 weeks depending on the technique) and they are costly. Finally, a number of chromosomal abnormalities go undetected.
Diagnosis of Pathologies with a Genetic Component
Many diseases have a recognized genetic component (diabetes, hypertension, obesity and the like) which is the result of deletions, insertions and/or chromosomal rearrangements of variable sizes. The culturing of cells does not pose any problem at this stage, but the FISH techniques, which are described by G. D. Lichter et al. (1993), Genomics, 16, 320-324; B. Brandritt, et al., (1991), Conomics, 10, 75-82 and G. Van den Hengh et al., (1992), Science, 257, 1410-1412) have a limited resolution and require a highly qualified staff, making these tests barely accessible.
The development of a more effective and inexpensive test would allow the general adoption of suitable therapies, at an early stage of the pathologies involved, likely to improve their remission.
Cancer Diagnosis
Among the pathologies with a genetic component, cancerous conditions constitute a major class affecting an increasing proportion of the population. Current understanding of the process of the onset of a cancerous condition involves a step of proliferation of proto-oncogenes (mutations in the genome of the cells) which precedes the transformation of the cell to a cancerous cell. This proliferation step is unfortunately not detectable, whereas the possibility of carrying out a treatment at this stage would certainly increase the chances of remission and would reduce the patients' handicap.
Finally, a number of tumors are characterized by chromosomal rearrangements such as translocations, deletions, partial or complete trisomies, and the like.
In each of these fields, molecular combing can provide a major contribution, either by the speed and the small quantity of biological material needed, or by the quantitative accuracy of the results.
The importance of the technique appears most particularly in the case where the genetic material is obtained from cells which are no longer dividing or which cannot be cultured, or even from dead cells in which the DNA is not significantly degraded.
In the case of prenatal diagnosis, such is the case after extraction of fetal cells circulating in the maternal blood (Cheung et al., 1996, Nature Genetics 14, 264-268). The same applies in the case of cancerous cells obtained from certain tumors.
Molecular combing makes it possible to improve the possibilities of diagnosis of genetic diseases, but it may also allow the study and identification of the genomic sequences responsible for said diseases. Moreover, currently, the development of a diagnostic “kit” or box starts with the search for the gene involved in the pathology.
The search for genes involved in pathologies (human or other) is nowadays generally carried out in several steps:
(i) Establishment of a target population of individuals effected by the pathologies, of their descendants, ascendants and collaterals, and collection of blood and/or cell samples for the purpose of storing genetic material (in the form of DNA or of cellular strains).
(ii) Genetic location by analysis of probability of cosegregation with genetic markers (linkage analysis). At this stage of the study, a few close markers located on one or more given chromosomes are available which make it possible to proceed to the step of physical location.
(iii) Physical mapping: starting with the genetic markers obtained in the preceding step, a screening of libraries of human DNA clones (YACs, BACs, cosmids and the like) specific for the region(s) determined in the precedi
Bensimon Aaron
Bensimon David
Michalet Xavier
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Horlick Kenneth R.
Institut Pasteur
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