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WO1988003572A1 - Sequence d'adn - Google Patents

Sequence d'adn Download PDF

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Publication number
WO1988003572A1
WO1988003572A1 PCT/GB1987/000807 GB8700807W WO8803572A1 WO 1988003572 A1 WO1988003572 A1 WO 1988003572A1 GB 8700807 W GB8700807 W GB 8700807W WO 8803572 A1 WO8803572 A1 WO 8803572A1
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WO
WIPO (PCT)
Prior art keywords
dna
sequence
probe
human
dmd
Prior art date
Application number
PCT/GB1987/000807
Other languages
English (en)
Inventor
Kay Elizabeth Davies
Terence James Smith
Lynn Wilson
Original Assignee
Kay Elizabeth Davies
Terence James Smith
Lynn Wilson
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kay Elizabeth Davies, Terence James Smith, Lynn Wilson filed Critical Kay Elizabeth Davies
Publication of WO1988003572A1 publication Critical patent/WO1988003572A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4707—Muscular dystrophy
    • C07K14/4708—Duchenne dystrophy
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • DMD DNA Sequence Duchenne muscular dystrophy
  • Boys affected by DMD suffer a progressive muscle wastage which may result in their being wheelchair bound by the age of 11. Life expectancy may be as little as 20 years.
  • a milder X-linked muscular dystrophy, Becker muscular dystrophy (BMD) shows clinical features which are similar to, but much less severe than, those of DMD. Some BMD sufferers may have a normal life span.
  • a DNA sequence has now been isolated from normal X chromosomal DNA which maps extremely close to,or within, the DMD locus, which sequence has been found to be deleted in the X chromosome of some boys suffering from DMD.
  • the present invention provides a DNA sequence characterized as follows:-
  • the DNA sequence having the above characteristics has the base sequence substantially as given in Figure 1.
  • HIP 25 the DNA sequence having this base sequence, to which we have given the designation HIP 25, has been deleted in 30 out of 404 DMD boys tested and in 3 out of 36 BMD boys tested.
  • HIP 25 the DNA sequence having this base sequence, to which we have given the designation HIP 25
  • HIP 25 has been deleted in 30 out of 404 DMD boys tested and in 3 out of 36 BMD boys tested.
  • This 15.5 kb sequence contains a unique 1 kb Hind III restriction fragment which fragment forms another embodiment of the DNA sequence of the invention.
  • a DNA probe which hybridises to, normal X- chroraosomal DNA but which does not hybridise to X- chromosomal DNA obtained from a patient indicates the deletion or mutation of the sequence complementary to the sequence of the probe from the DNA of the patient.
  • the DNA sequence of the invention can be utilized in the form of a DNA probe for the detection of a deletion or a mutation of that sequence from the DNA of an individual.
  • the present invention further provides a DNA probe comprising a single stranded DNA molecule capable of hybridising to the DNA sequence of the invention or its complementary sequence or a fragment of either of these DNA sequences, which single stranded DNA molecule is labelled with a detectable marker.
  • the DNA probe is selected from the group consisting of the sequence designated HIP 25 and its complementary sequence and fragments of these, ' labelled with a detectable marker.
  • the DNA probe is a 1 kb Hind III restriction fragment, its complementary DNA sequence, or subfragment thereof labelled with a detectable marker.
  • the detectable marker will be selected from fluorescent and radioactive markers. Such markers are well known in the art and need not be described in detail.
  • the present invention provides a method of screening human X chromosomes for the detection of the DMD mutational sites which method comprises treating genomic DNA obtained from a patient with the DNA probe of the invention under hybridising conditions and then analysing the treated DNA to detect the presence or absence of the labelled marker and comparing the result obtained with the result obtained using genomic DNA from a normal human.
  • One suitable way of carrying out such a method would be to employ the well-known Southern blotting technique. According to this technique, the DNA to be treated is cut with one or more restriction enzymes and the restriction fragments are separated according to size by gel electrophoresis.
  • the DNA in the gel is transferred to a nylon or nitrocellulose filter which is then hybridised with the labelled probe which binds to any DNA fragments containing a DNA sequence complementary to that of the probe. Any bound probe is then detected by means of its detectable marker.
  • a positive result for the detection of the labelled probe bound to the DNA fragment identifies the DNA sequence as being present in the X-chromosome.
  • a negative result indicates that the DNA sequence tested for is deleted from the .patients X- chromosome.
  • HIP 25 The DNA sequence designated HIP 25 was isolated by the following procedure. .A sample of DNA derived from a patient having a deletion at Xp 21-2 and who suffers from DMD, chronic granulomatous disease (CGD), retinitis pigmentosa (RP) and also McLeod's syndrome was' sheared by sonication and denatured by techniques well known in the art. DNA from an X-library was grown up in bulk and was then treated with the enzyme EcoRI followed by treatment with the enzyme Sau 3AI.
  • the enzyme-cleaved DNA from the X-library was then mixed with the sheared, denatured DNA from the patient under reassociation conditions in the presence of a buffer comprising disodium hydrogen phosphate and sodium dihydrogen phosphate and left for a period of time to allow reassociation of DNA segments.
  • the DNA was then purified.
  • the DNA obtained from the reassociation reaction was ligated into Bam Hi-cleaved plasmid DNA to form recombinant plasmids which were transformed into a suitable host and cloned to select for those having DNA inserts mapping within the Xp 21.2 deletion.
  • the DNA used was from a cell line derived from a patient, BB, who has a deletion of Xp 21.2 and suffers from DMD, CGD and RP and the McLeod Syndrome.
  • the DNA was sheared by sonication using a DAWE SONIPROBE to a mean size of 500 - 1000 base pairs.
  • the X-library DNA was cleaved with EcoRI (2U ⁇ g ⁇ 1 DNA in 100mM Tris-HCl pH 7.5, 50mM NaCl, 5mM M Cl 2 ) and the restriction fragments obtained, purified by low m.p.
  • the reassociated DNA obtained was inserted into Bam Hi-cut plas id DNA (pUC 18).
  • the cleavage conditions with Bam HI were:- 2U units ⁇ g ⁇ DNA in 150mM NaCl, 6mM Tris-HCl pH 7.9, 6mM M Cl 2 .
  • the recombinant plasmlds obtained above were introduced into E. coli JM 101 cells and the transformed bacteria were plated on L broth.
  • the resulting library was assayed for clones containing plasmid DNA with the required insert.
  • the plas ids were- digested with the restriction enzyme Pvu II under standard conditions to select those giving restriction fragments of suitable length.
  • the cleaved DNA sample was labelled with 32P, using a technique well- known in the art, for use in the hybridisation.
  • the filters, after hybridisation, were washed at 1 x SSC (0.15 mM NaCl, 0.015 M Na citrate), pH 7.5 at 65°C.
  • Autoradiography was carried out at a temperature of - 70°C for 3 days. Results are -given in Table I.
  • hybrid cell line containing Xpter-Xqter (different hybrid from that used in lane 6. + LANE No. DNA source Hybridization
  • LANE No. DNA source Hybridisation (X chromosome) ( + or - ) to
  • the DMD patients 1 to 4 previously found to be deleted for the pERT 87 and the XJ 1.1 loci L.M. Kunkel et al, NATURE, 322_, 73-77 (1986)), were found to be deleted for the DNA sequence of HIP 25.
  • pERT 84 which lies centromeric from pERT 87
  • a positive signal was seen for all of the samples except for that in lane 3-
  • the centromeric breakpoint of the deletions in DMD patients 1 , 3 and 4 lies between pERT 84 and HIP 25 but, in the case of DMD patient 2, the breakpoint must lie between the loci 754 and pERT 84 in the X chromosome.
  • the order of sequences on the short arm of the human X chromosome relative to the HIP 25 is believed to be as follows:-
  • RFLP restriction fragment length polymorphism
  • yeast t. RNA 5 pg
  • the DNA sequence of the invention under low stringency conditions hybridises strongly to mRNA from several tissue types (A).
  • A hybridises strongly to mRNA from several tissue types
  • A hybridises strongly to mRNA from several tissue types
  • A hybridises strongly to mRNA from several tissue types
  • A hybridises strongly to mRNA from several tissue types
  • A hybridises strongly to mRNA from several tissue types
  • the DNA sequence of the invention did not hybridise to mRNA derived from fetal fibroblasts.
  • the results obtained, together with the sequence conservation noted in EXPERIMENT 4 suggests that the DNA sequence of the invention plays an important role in muscle function.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On a localisé une séquence d'ADN qui est supprimée chez un certain nombre de patients atteints de distrophie musculaire de Duchenne (DMD). Cette séquence s'hybride fortement avec de l'ARNm de muscle foetal humain mais seulement faiblement avec de l'ARNm de muscle adulte humain ou de rat. Elle ne s'hybride pas avec de l'ARNm dérivé de fibroblastes foetaux humains. La séquence d'ADN ou une partie de celle-ci peut s'utiliser comme sonde d'hybridation lorsque l'on examine l'ADN chromosomique X de patients en vue d'y détecter la présence de sites mutationnels de DMD.
PCT/GB1987/000807 1986-11-13 1987-11-12 Sequence d'adn WO1988003572A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8627123 1986-11-13
GB868627123A GB8627123D0 (en) 1986-11-13 1986-11-13 Dna sequence

Publications (1)

Publication Number Publication Date
WO1988003572A1 true WO1988003572A1 (fr) 1988-05-19

Family

ID=10607267

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1987/000807 WO1988003572A1 (fr) 1986-11-13 1987-11-12 Sequence d'adn

Country Status (2)

Country Link
GB (1) GB8627123D0 (fr)
WO (1) WO1988003572A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989006286A3 (fr) * 1987-12-22 1989-08-10 Childrens Medical Center Procedes et sondes de diagnostic de dystrophie musculaire
WO1990005194A1 (fr) * 1988-11-01 1990-05-17 Medical Research Council Sonde permettant de detecter la presence d'un syndrome x fragile
WO1991009140A1 (fr) * 1989-12-12 1991-06-27 Medical Research Council Sonde de detection du syndrome de l'x fragile
WO1994000597A1 (fr) * 1992-06-23 1994-01-06 Pharmacia Lkb Biotechnology Ab Procede et systeme de diagnostics biologiques moleculaires

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
NATURE, Volume 316, 29 August 1985, A.P. MONACO et al., "Detection of Deletions Spanning the Duchenne Muscular Dystrophy Locus Using a Tightly Linked DNA Segment", pages 842-845. *
NATURE, Volume 318, 19/26 December 1985, P.N. RAY et al., "Cloning of the Breakpoint of an X;21 Translocation Associated With Duchenne Muscular Dystrophy", pages 672-675. *
NATURE, Volume 322, 3 July 1986, P.N. GOODFELLOW, "Duchenne Muscular Dystrophy. Collaboration and Progress", pages 12-13. *
NATURE, Volume 323, No. 6089, 16 October 1986, A.P. MONACO et al., "Isolation of Candidate cDNAs for Portions of the Duchenne Muscular Dystrophy Gene", pages 646-650. *
NUCLEIC ACIDS RESEARCH, Volume 15, No. 5, 11 March 1987, IRL PRESS LTD., (Oxford, GB), T.J. SMITH et al., "Isolation of a Conserved Sequence Deleted in Duchenne Muscular Dystrophy Patients", pages 2167-2174. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989006286A3 (fr) * 1987-12-22 1989-08-10 Childrens Medical Center Procedes et sondes de diagnostic de dystrophie musculaire
WO1990005194A1 (fr) * 1988-11-01 1990-05-17 Medical Research Council Sonde permettant de detecter la presence d'un syndrome x fragile
WO1991009140A1 (fr) * 1989-12-12 1991-06-27 Medical Research Council Sonde de detection du syndrome de l'x fragile
WO1994000597A1 (fr) * 1992-06-23 1994-01-06 Pharmacia Lkb Biotechnology Ab Procede et systeme de diagnostics biologiques moleculaires

Also Published As

Publication number Publication date
GB8627123D0 (en) 1986-12-10

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