WO2000024925A1 - Moyen et procede de caryotypage - Google Patents

Moyen et procede de caryotypage Download PDF

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WO2000024925A1
WO2000024925A1 PCT/AU1999/000938 AU9900938W WO0024925A1 WO 2000024925 A1 WO2000024925 A1 WO 2000024925A1 AU 9900938 W AU9900938 W AU 9900938W WO 0024925 A1 WO0024925 A1 WO 0024925A1
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unknown
dna
chromosomal
karyotype
chromosome
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PCT/AU1999/000938
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Nicole Hussey
Colin Matthews
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Luminis Pty Ltd
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Priority to AU12540/00A priority Critical patent/AU1254000A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • This invention relates to determining differences in chromosomes between individuals.
  • Genetic screening for abnormalities that give rise to adverse health consequences is a well established procedure that may be used in prenatal diagnosis of abnormalities.
  • the location of specific genes and/or diagnostic markers can be identified from portions of the genome that are present in abnormal copy numbers.
  • foetal cells needed to be cultured for several weeks to obtain enough cells for analysis.
  • techniques of in situ hybridisation that allow analysis of intact cell nuclei have been developed.
  • PCR Polymerase chain reaction
  • FISH fluorescent in situ hybridisation
  • Comparative genomic hybridisation is a technique that has been used for determining the gains and losses of copy number of chromosomes or partial chromosomes from subject cells without chromosome preparation from test tissue (US patent 5665549 to Pinkel et al). CGH is now mainly used for the study of the genetics of cancer (Kallioniemi et al , 1996).
  • a single cell contains less than 25 pg genomic DNA (Kuukasjarvi et al:, 1997) and thus substantially the whole genomic DNA from a single cell has to be amplified before it is used for a CGH test.
  • DOP-PCR degenerate oligonucleotide primed
  • the present invention results from the finding that it is possible to reliably perform CGH karyotyping using DNA amplified from single cells.
  • the invention could be said to reside in a method of comparing at least one chromosome or part thereof from a cell of an individual with an unknown karyotype with the corresponding chromosome or part thereof of an individual with a known karyotype, the method including the steps of : a. obtaining DNA derived from a single cell of the unknown karyotype, b . amplifying the chromosomal DNA of the unknown karyotype sufficiently for comparative purposes, c. labelling the amplified unknown chromosomal DNA with a first label and labelling a known chromosomal DNA with a second label, the first and second label being detectably different, d .
  • hybridising the labelled known chromosomal DNA with a chromosomal spread and hybridising the labelled and amplified unknown chromosomal DNA with the chromosomal spread, and e. mapping the chromosomal spread, and comparing the relative amount of first and second label as a function of position on the at least one chromosome or part thereof.
  • the known chromosomal DNA is also obtained from a single cell source of known karyotype, and amplified before labelling in step c.
  • One of the preferred applications of this invention is in the prenatal diagnosis of chromosome differences for which application the source of the unknown single cells might thus be one of a number of types.
  • the source of the unknown single cells might thus be one of a number of types.
  • an embryo it could be a single embryo cell which has been removed from a pre-implantation embryo by micromanipulation. It is known that normal births can result after cells are taken from such embryos. It will of course be understood that the amount of sample material taken from an embryo is very much limited.
  • Other sources for prenatal testing might be oocyte, polar body, sperm cells or other somatic or germ cells.
  • the sample might be a foetal blood cell taken from the maternal circulation or from the mothers reproductive tract (eg cervical or vaginal lavage).
  • Foetal red blood cells unlike mature blood cells, are nucleated and can be isolated in sufficient numbers from the maternal circulation to provide a reliable source of whole chromosomal DNA for testing purposes. This source is preferable to performing invasive procedures such as amniocentesis, which have a risk factor associated with them.
  • a single cell as a source of the DNA of known karyotype and this may be chosen from a source easily collected such as white blood cells, but other sources might also readily be used. It has been found that a single cell of known karyotype may give clearer results and greater coverage of the genome than may be obtained using DNA from multiple cells and or other sources of DNA of known karyotype.
  • a similar effect may also be obtained using a small number of cells, and in particular if the number of cells of known and unknown are matched, perhaps to the same order of magnitude, and any DNA is isolated and amplified to approximately the same degree. It will be understood that using the technique of micromanipulation it is possible typically to isolate a single cell however it may be possible to obtain two or more cells and therefore the invention also contemplates the use of a small number of cells that may be obtained by micromanipulation. Thus, reference throughout this specification to a 'single cell' is to be understood to include a small number of cells, perhaps less 10 cells, and more preferably less than 5 cells.
  • the known chromosomal DNA and the unknown chromosomal DNA are amplified by PCR using the same complement of primers for both.
  • the quality and quantity of the extension products resulting from the PCR reaction of both the known and unknown DNA are comparable.
  • the results obtained using the present method are to be contrasted to those obtained if a tissue extract is used as a control and labelled, perhaps by nick translation, so that the DNA used is qualitatively different.
  • the distribution of binding of primers to the chromosomes of the cell of known and unknown karyotypes means that after one round of extension by PCR the result is not two copies of the genome concerned, that is, the extension products resulting from PCR do not meet end to end to collectively form a complete genome.
  • the amplification of known and unknown DNA is by means of using degenerate primers, the technique sometimes being referred to as degenerate oligonucleotide primed (DOP)-PCR (Telenuis et al, 1992), which reference is incorporated herein in its entirety.
  • DOP degenerate oligonucleotide primed
  • This technique uses a family of primers, each with identical flanking sequences and a "random" central portion.
  • Use of DOP-PCR generated extension products is known to give an adequate distribution of PCR extension products for purposes of CGH in tissue samples.
  • the DOP-PCR technique can also be used to label the extension products appropriately by the incorporation of a labelled nucleotide into the reaction mix in the final round or rounds of amplification.
  • primer for the DOP-PCR need not be the commonly used degenerate universal primer but could also be any primer that amplifies a proportion of the genome sufficient to allow for the comparative analysis. Indeed other forms of so-called whole cell amplification might equally be used.
  • PEP-PCR primer-extension preamplication PCR
  • ligation mediated PCR Klein et al, 1999
  • tagged PCR t-PCR; Grothues et al, 1993; incorporated herein in its entirety
  • alu-PCR Nelson et al, 1989; incorporated herein in its entirety
  • the chromosomal spread is in the form of a metaphase spread slide from a known normal male.
  • the mapping may involve forming an image of the chromosomal spread and comparing the relative amount of the first and second label as a function of position on the chromosome part thereof.
  • each known and unknown amplified and labelled DNA preparation is hybridised to more than one chromosome spread to minimise the effects of any lack of uniformity of hybridisation that might occur on any given metaphase spread.
  • preferably greater than 90 % of the distributions of the standard deviations in autosomal regions fall within the range 0.85 - 1.15, the variation limits of the profile in a successful CGH (Kallioniemi et al, 1994).
  • genomic DNA was the reference material about 30 % of the distributions of the standard deviations in autosomal regions fell outside the range 0.85 - 1.15.
  • the chromosomal spread is in the form of an array of different nucleic acids representative of a complement of chromosomes and attached to the surface of a substrate (a 'gene chip').
  • the labelled and amplified unknown and known chromosomal DNA may be hybridised with the biological plate of the gene chip, and the chromosomal spread mapped by interrogating arrays on the biological chip plate with a biological chip plate reader to generate assay results.
  • mapping of the chromosome spread may include forming an image with intensities in each region of the image varying according to the relative amount of first and second label.
  • heterochromatic or telomeric regions are generally referred to as heterochromatic or telomeric (end of chromosomes) regions. Often heterochromatin is associated with centromeres and thus centromeric regions cannot be analysed by CGH and these regions are discounted when comparisons of the binding between known and unknown karyotypes are made.
  • the invention preferably involves an assessment being made as to the number of copies of a chromosome(s) or portion thereof between individuals of known and unknown karyotype.
  • This technique is particularly useful in diagnosis of gross chromosomal differences and aneuploidy such as deletions, duplications or other amplifications.
  • Conditions that might be amenable to detection by this technique include but are not limited to Trisomy 21, 13, 18 and detecting missing chromosome such as in Turner's syndrome (45, XO). Whilst it will be understood that for the purpose of scanning for these general defects it is desirable to look at all chromosomes of the subject, it might under certain circumstances be desirable to look at only a partial karyotype, and the chromosome spread or gene chip need not necessarily include all chromosomes.
  • the invention might be also be said to have resulted from the finding that the combination of DOP-PCR and CGH gives an effective result, whether a single cell source of DNA of unknown karyotype is used or not.
  • the CGH analysis will permit very fine analysis but only for rearrangements that are in the order of magnitude of mega bases, however, it may be possible to use the same sample of amplified DNA for testing for the presence or absence of specific alleles or defects in a gene.
  • a specific primer or primer group can be added to amplify the unknown DNA sample (and known sample) the primer or primer set might not permit amplification of a region of the chromosome with the specific defect or allele and then the absence of an allele can be probed for by a suitable probe, with suitable controls.
  • chromosomes 21 and 22 are quite small in size and any chromosomal variation is relatively difficult to visualize especially in view of the fact that both these chromosomes have heterochromatic regions. It might be desirable to bias the primer complement in favour of highlighting certain regions, and to add primers specific for that region or chromosome. Thus highlight primers designed to highlight certain chromosomes or portions of chromosomes might be added to the complement of primers. Those highlight primers might be made to bind to sequences known to be present in certain positions of the target for highlighting.
  • chromosome 21 markers such as D21S131, and D21S142 and similar markers might be used, or in the alternative DNA sequences for any chromosome might be determined from the data resulting from the cooperative project for DNA sequencing of the entire human genome known as HUGO.
  • the invention could be said to reside in a method of comparing at least one chromosome or part thereof from a cell of an individual with an unknown karyotype with the corresponding chromosome or part thereof of an individual with a known karyotype, the method including the steps of : a. obtaining DNA derived from a single cell of the unknown karyotype, b . amplifying the chromosomal DNA of the unknown karyotype sufficiently for comparative purposes using: a degenerate primer for amplifying substantially the whole genome, and at least one specific highlight primer or primer group for amplifying specific loci or loci groups, c .
  • the method of the second aspect of the invention therefore allows for the detection of general chromosomal abnormalities as well as allele differences for specific loci.
  • Candidate genes to amplify with specific primers in addition to the general (DOP or similar) primers include disease loci of interest.
  • disease loci for example, Caucasians have a high incidence of cystic fibrosis (CF) therefore relevant primers for this locus could be included.
  • Other disease loci for example include thalassemia, Duchenne muscular dystrophy, rare X-linked disorders, Haemophilia, Huntington's Disease. Indeed the molecular defect(s) behind single gene disorders and multiple gene disorders are being elucidated rapidly in this the genetic era. Therefore the number and type of primers added will increasingly reflect a specific mix determined to be beneficial to each family.
  • other loci may also be amplified specifically such as polymorphic loci. This enables paternity/ maternity testing and the detection of contamination to increase the confidence in diagnosis.
  • Obtention and amplification of DNA derived from cells of known and unknown karyotype, labelling of the amplified unknown chromosomal DNA with a first label and labelling a known chromosomal DNA with a second label, hybridisation of the labelled known and unknown chromosomal DNA with the chromosomal spread, and comparing the relative amount of first and second label may be performed according to the first aspect of the invention.
  • Figure 1 is a photograph of various DNA samples eletrophoresed on an agarose gel and stained with ethidium bromide. The following were loaded in each lane:- Lane 1: SSP-1 DNA/Eco RI fragment: 360-8510 bp, Lane 2- 4: typical DOP-PCR products derived from 3 individual single cells,
  • Lane 7 DNA/Hind III fragment: 125 - 23,130 bp
  • Lane 8 pUC19DNA/Hpa II fragment: 26 - 501 bp
  • Figure 2 is a graphic representation printed by the Quips CGH
  • a graphic of the chromosome is represented to the left.
  • the centromere of each chromosome is indicated by a constriction, the dark bands are representative of bands seen in staining of chromosomes and cross hatched portions represent heterochromatin portions of the chromosome concerned.
  • a thick vertical line is some times present as a complete or broken line immediately adjacent to the graphic of the chromosome indicating when a region of one or other of the chromosomes has been calculated to have at least one further copy of a chromosome or region thereof.
  • the vertically aligned graph to the right includes a central medial line, with darkened vertical lines to either side of the centre and a further three lines progressively further from the darkened lines.
  • a calculation of the extent to which any region is increased or decreased in either the known or unknown chromosome is graphed and indicated by the dark graphed line, the standard deviation is also graphed in lighter lines on either side of the dark graphed lines. Once the dark graphed line breaks past the first of the vertical lines of the centre it is considered that an increase or decrease has occurred with respect to the known cell, and
  • Figure 3 is a representation similar to that of Figure 2 showing the results of the CGH of amplified DNA-SG (47 XY, +13) against amplified normal genomic DNA-TRITC (46, XY)
  • Figure 4 is a representation similar to that of Figure 3 showing the results of the CGH of amplified DNA-SG (47 XY, +21) against amplified normal genomic DNA-TRITC (46,
  • Figure 5 is a representation of a microarray showing that a single cell tested relative to a known normal cell had extra copies of the terminal portion of chromosome 18 (as indicated by green fluorescence v£/ ), a deletion of the terminal portion of chromosome 14 (as indicated by red fluorescence ⁇ ) and normal chromosome 21 constitution (as indicated by yellow fluorescence J ), and
  • Figure 6 is a photograph of various DNA samples eletrophoresed on an agarose gel and stained with ethidium bromide. The following were loaded in each lane:- Lane 1: phage Lambda/Hind III fragments: 125 - 23,130 bp, Lane 2: pUC19 Hpa II fragments: 26-501 bp,
  • Lane 3 DOP-PCR products derived from an individual single cell amplified without the addition of specific primer for exon 10 of the cystic fibrosis gene
  • Lane 4 DOP-PCR products derived from an individual single cell amplified with the addition of specific primer for exon 10 of the cystic fibrosis gene
  • a single cell each from an individual with the karyotype of 46,XY, 46,XX, 45,XO, 47,XXY, 47,XY,+13 and 47,XY,+21 were sorted into a 0.5 ml PCR tube under an inverted microscope.
  • 5 ⁇ l of 200mM KOH, 50 mM DTT solution was added to each tube and heated at 65°C for 10 minutes for cell lysis.
  • an equal amount (5 ⁇ l) of 900mM Tris-HCl pH 8.3, 300 mM KCl, 200 mM HC1 was added to neutralise the buffer.
  • DOP-PCR was run on a thermocycler (Minicyler, MJ Research) with the initial denaturation at 94 °C for 10 min, 5 cycles of random amplification: 94 °C for 1 min, 30 °C for 1.5 min, ramping from 30-72 °C for 3 min, 72 °C for 3 min, 35 cycles of non- random amplification: 94 °C for 1 min, 62 °C for 1 min, 72 °C for 3 min + 14 sec/cycle.
  • the size of DOP-PCR product was checked by electrophoresis through a 1 % agarose gel. DOP-PCR products were purified using a High Pure PCR Purification Product kit (Boehringer Mannheim) and DNA concentration was estimated spectrophotometrically.
  • DOP-PCR labelling The amplified single cell DNA was labelled with SpectrumGreen (SG)-dUTP (Vysis) by another round of DOP-PCR.
  • the reaction solution of labelling DOP-PCR (Ghaffari et ah, 1998) was comprised of 2 ⁇ l of amplified DOP-PCR products, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.0 mM MgCl , 0.001 % gelatine, 2 ⁇ M degenerated universal primer, 0.2 mM of dATP, dCTP, dGTP, 0.05 mM dTTP and 0.15 mM SpectrumGreen-dUTP (Vysis). 2.5 U of Taq polymerase.
  • the supernatant was discarded and the pellet was dried for a few minutes at 60°C and dissolved in 10 ⁇ l 50% formamide, 10 % dextran sulphate, 2 x SSC, pH 7.0.
  • the DNA was denatured at 70°C for 5 min just before hybridization.
  • a routinely prepared metaphase spread slide from a known normal male was dehydrated through a 70 %, 95 % and 100 % ethanol series.
  • the slide was denatured in 70% deionized formamide, 2 x SSC, pH 7.0 at 70°C for 3 min.
  • the denatured DNA mixture was applied onto the denatured chromosomes on the slide under a 18 x 18 mm coverslip sealed with rubber cement and incubated in a wet chamber at 37°C for 2-3 days.
  • the posthybridization washing was carried out in 0.4 % NP40 (NONIDET®P40), 0.3 x SSC and 0.1 % NP40, 2 x SSC for 2 min each at 70 °C. After air-drying, the preparation was counterstained with an antifade-mounting solution, 0.56 ⁇ g/ml DAPI in VectaShield (Vector Laboratories).
  • CGH images were captured by a CCD camera using the Quips XL Genetics Workstation (Vysis 30-143200) or Cytovision Ultr Image Collection System ( Applied Imaging Int Ltd) and CGH analyses were carried out using Quips CGH Analysis Software (Vysis 30-143002).
  • the yield of a single cell DOP-PCR reached at least 2 ⁇ g DNA as determined by spectrophotometric measurement after purification.
  • the size of the amplified product ranged from 200 to 23,000 bp (predominantly : 400- 4000).
  • the negative control always contained primer related products but their size was usually much shorter (about 1,000 bp) than single cell DOP-PCR product.
  • the size of nick translation labelled DOP-PCR product was 100 - 1,500 bp.
  • the profile for a cell (47, XY +13) containing an extra chromosome 13 clearly demonstrates an increase in the amount of Green fluorescence confirming the extra chromosome 13 material present in this unknown cell relative to the single male (known normal) cell reference DNA.
  • EXAMPLE 2 Whole genome amplification and CGH using DOP-PCR and biological chip detection
  • the traditional karyotype of an individual involves growing up around 10 7 cells from whole blood to give an actively dividing population of cells. The cells are then arrested in mitosis and spread onto a microscope slide to give a so called "metaphase spread". Metaphase chromosomes are sufficiently dense to be able to be seen and analysed by light microscopy.
  • DNA chip technology it is no longer necessary to use traditional metaphase spreads for karyotyping. Instead an array of DNA (or RNA) clones or PCR products can be made on the traditional microscope slide.
  • DNA (or RNA) clones or PCR products can be made on the traditional microscope slide.
  • One, thousands and eventually hundreds of thousands of 2 nano litre spots (clones or PCR products) can be placed onto a single slide.
  • spots correlate to regions of the genomes in a defined manner such that it is known what position and along which chromosome the spot corresponds to in much the same way that a traditional karyotype does. It can be thought of that the old fashioned metaphase spread is an "analogue" version of a karyotype and that this is being replaced by DNA chips which are a "digital” version of a karyotype. The later being more amenable to automation and computer analysis.
  • Clones can be bought, made from scratch or obtained from Whole Chromosome Paint (WCP) Probes which are commercially available (eg Vysis). To adapt WCP probes for this purpose it is necessary to isolate just certain of the individual probes within a WCP probe. This can be achieved by serial dilution or other means of picking clones both randomly and nonrandomly.
  • WCP Whole Chromosome Paint
  • FIG. 5 A typical result using a micro array is shown in Figure 5. This result indicates that the single cell tested here relative to a known normal cell had extra copies of the terminal portion of chromosome 18 (as indicated by green fluorescence), a deletion of the terminal portion of chromosome 14 (as indicated by red fluorescence) and normal chromosome 21 constitution (as indicated by yellow fluorescence).
  • An aliquot of the PCR reaction can be used to investigate specific loci of interest by other means not utilising gene chips or by a method that enables analysis by the same or a different gene chip (as appropriate) to that performing the CGH analysis.
  • the invention described here allows the "karyotypic" information to be combined with more conventional diagnoses for single gene disorders. In particular it allows the amplification with high efficiency of multiple other loci from a single cell to be combined with CGH analysis.
  • Candidate genes to amplify with specific primers in addition to the general (DOP or similar) primers include disease loci of interest.
  • disease loci of interest For example cystic fibrosis (CF), thalassemia, Duchenne muscular dystrophy, rare X-linked disorders, Haemophilia, Huntington's Disease.
  • CF cystic fibrosis
  • Duchenne muscular dystrophy rare X-linked disorders
  • Haemophilia Huntington's Disease.
  • any molecular marker which is determined to be a causative agent of a genetic disorder or merely to correlate with the disease in general or in that particular family can be used. Therefore the number and type of primers added will increasingly reflect a specific mix determined to be beneficial to each family.
  • disease loci it is also important to amplify other loci specifically such as polymorphic loci. This enables paternity/ maternity testing and the detection of contamination to give increased confidence in diagnosis.
  • EXAMPLE 4 Whole genome amplification using PEP (primer extension preamplification) and specific loci amplification
  • CFTR Gene PCR amplification of exon 11 of the CFTR gene was performed as follows. 2.5 ⁇ l SEP aliquots were added to exon 11 mastermix (47.5 ⁇ l) containing 10 pmol forward primer, 10 pmol reverse primer (5'-CAACTGTGGTAAAGCAATAGTGT-3'; 5'- GCACAGATTCTGAGTAACCATAAT-3'), IX PCR buffer [50 mM KCl, 10 mM Tris.HCl, pH 8.3], 100 ⁇ M each dNTP, 2.5 mM MgCl and 1U Taq polymerase [Perkin Elmer] .
  • Tubes were placed into a pre-heated 96 °C MJ Research PTC- 100 PCR machine with hot bonnet and after an initial denaturation step of 94°C for 5 min, cycling conditions were 94°C for 30 sec, 60 °C for 60 sec, 72 °C for 45 sec for 35 cycles.
  • PCR amplification of exon 4 and 10 was the same as for exon 11 except that 200 ⁇ M each dNTP was used instead of 100 ⁇ M and primers were for exon 4 5'-
  • PCR amplification of exon 20 of the CFTR gene followed the procedure of Avner et al, (1994). Briefly, 2.5 ⁇ l SEP aliquots were added to mastermix (47.5 ⁇ l) containing 10 pmol forward primer, 10 pmol reverse primer (5'-TACCTATATGTCACAGAAGT- 3'; 5'-GTACAAGTATCAAATAGCAG-3'), IX PCR buffer [50 mM KCl, 10 mM Tris.HCl, pH 8.3], 200 ⁇ M each dNTP, 1.5 mM MgCl and 1U Taq polymerase [Perkin Elmer]).
  • Tubes were placed into a pre-heated 96 °C MJ Research PTC- 100 PCR machine and after an initial denaturation step of 94°C for 5 min, cycling conditions were 94°C for 30 sec, 50 °C for 30 sec, 72 °C for 30 sec for 35 cycles.
  • Amplification of the SRY gene was a modification of the method of Cui et al, (1994).
  • SRY PCR mix (IX PCR buffer [50 mM KCl, 10 mM Tris.HCl, pH 8.3], 375 ⁇ M each dNTP, 3.75 mM MgCl, 10 pmol forward primer (5'-CATGAACGCATTCATCGTGTGGTC-3'; 5'- CTGCGGGAAGCAAACTGCAATTCTT-3'), 10 pmol reverse primer and 1U Taq polymerase [Perkin Elmer]).
  • Tubes were placed into a pre-heated 96°C Corbett FTS- 100 PCR machine and after an initial denaturation step of 94°C for 5 min, 35 cycles of 94°C for 1 min, 65°C for 1 min, 72°C for 2 min were carried out followed by a final extension step of 72°C for 10 min.
  • PCR products were electrophoresed on 2% agarose gels prestained with ethidium bromide.
  • SEP was also performed using Duchenne muscular dystrophy (DMD) as the disease example.
  • DMD Duchenne muscular dystrophy
  • the SEP reaction was performed as described for the CFTR gene except that the DMD gene primers for exons 17, 19, 44, 45, 48 and exon 8 (Hussey et al., in press, and references cited therein) were used instead of the CF gene specific primers. All other experimental details were the same.
  • the gel showed the presence of bands in all locus specific reactions (exons 8, 17, 19, 44, 45, 48 from the DMD gene and the SRY gene) and consistent intensities between single cells especially compared to the positive controls.
  • One of the benefits flowing from this invention include the fact that karyotyping can be achieved with only minute samples.
  • a single cell can be used as the basis of a DNA sample for testing of chromosomal abnormalities and any specific gene defects of interest for the purposes of prenatal diagnosis and/ or Preimplantation Genetic Diagnosis (PGD).
  • PPD Preimplantation Genetic Diagnosis

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Abstract

L'invention concerne un procédé de comparaison d'au moins un chromosome ou d'une partie de celui-ci, provenant d'une cellule d'un individu dont le caryotype n'est pas connu, à un caryotype connu. Ledit procédé consiste à: (a) obtenir l'ADN dérivé d'une seule cellule dudit caryotype inconnu ; (b) amplifier l'ADN chromosomique du caryotype inconnu, suffisamment pour permettre la comparaison; (c) marquer l'ADN chromosomique inconnu amplifié à l'aide d'un premier marqueur et marquer un ADN chromosomique connu à l'aide d'un deuxième marqueur, les premier et deuxième marqueurs étant différents à la détection; (d) hybrider l'ADN chromosomique connu avec une bande chromosomique, et hybrider l'ADN chromosomique inconnu amplifié à la bande chromosomique; (e) former une image de la bande chromosomique et comparer la quantité relative des premier et deuxième marqueurs en fonction de la position sur le ou les chromosomes ou une partie de ceux-ci. Il est également, possible dans la partie (b), d'ajouter des amorces supplémentaires pour la mise en valeur de locus spécifiques à étudier. Le procédé peut être utilisé pour le diagnostic de préimplantation à partir d''une seule cellule source.
PCT/AU1999/000938 1998-10-28 1999-10-28 Moyen et procede de caryotypage WO2000024925A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002044411A1 (fr) * 2000-12-01 2002-06-06 Rosetta Inpharmatics, Inc. Utilisation de profils dans la detection de l'aneuploidie
DE10242359A1 (de) * 2002-09-12 2004-03-25 Alopex Gmbh Verfahren zur Amplifikation genetischer Informationen
WO2004088310A1 (fr) * 2003-04-02 2004-10-14 Adelaide Research & Innovation Pty Ltd Hybridation genomique comparative
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WO2002044411A1 (fr) * 2000-12-01 2002-06-06 Rosetta Inpharmatics, Inc. Utilisation de profils dans la detection de l'aneuploidie
DE10242359A1 (de) * 2002-09-12 2004-03-25 Alopex Gmbh Verfahren zur Amplifikation genetischer Informationen
WO2004027089A1 (fr) * 2002-09-12 2004-04-01 Alopex Gmbh Procede d'amplification d'informations genetiques
JP2006506054A (ja) * 2002-09-12 2006-02-23 アロペクス ゲーエムベーハー 遺伝情報の増幅のための方法
WO2004088310A1 (fr) * 2003-04-02 2004-10-14 Adelaide Research & Innovation Pty Ltd Hybridation genomique comparative
US8211642B2 (en) 2003-04-02 2012-07-03 Adelaide Research & Innovation Pty Ltd Comparative genomic hybridization
EP1678329A2 (fr) * 2003-10-30 2006-07-12 Tufts-New England Medical Center Diagnostic prenatal a l'aide d'adn foetal acellulaire dans le liquide amniotique
EP1678329A4 (fr) * 2003-10-30 2008-07-02 Tufts New England Medical Ct Diagnostic prenatal a l'aide d'adn foetal acellulaire dans le liquide amniotique
WO2006010610A2 (fr) * 2004-07-27 2006-02-02 Alopex Gmbh Procede permettant de determiner la frequence de sequences dans un echantillon
WO2006010610A3 (fr) * 2004-07-27 2006-06-22 Alopex Gmbh Procede permettant de determiner la frequence de sequences dans un echantillon
WO2011138750A1 (fr) * 2010-05-05 2011-11-10 Medical Research Council Procédé de comptage des nombres de copies de chromatides dans une cellule individuelle

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