Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-01-24
1998-04-07
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 536 231, 536 243, 536 2433, C12Q 168, C12P 1934, C07H 2104, C07H 2102
Patent
active
057363234
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The cloning of cDNAs corresponding to the gene coding for the human angiotensin converting enzyme (ACE) has made it possible to use it as a DNA probe for investigating DNA polymorphism in this gene in man.
The interest of this research in DNA polymorphisms was multiple. On the one hand, it was known that the plasma level of the ACE was stable and the effect of an allele on a major gene had been suggested following a familial study. It was thus logical to want to know whether the ACE gene itself was responsible for the effect of the major gene and to discover the importance of the interpersonal differences of the ACE level as a function of the genotype. On the other hand, it was of interest to have available a genetic marker which could be used to test the involvement of the gene in certain diseases, such as hypertension, by association or binding studies.
From the clinical point of view, the plasma level of the ACE was measured in 80 normal individuals and their genotypes were determined. Significant differences were observed between the ACE level of the individuals as a function of their ACE genotype (Rigat, B. et al. (1990), Journal of Clinical Investigation, 1343-1346).
The research into polymorphism was conducted by using the Southern blot method. The DNA of different individuals was digested by several restriction enzymes. After hybridization with the cDNA probe for ACE, it was apparent that some fragments did not have the same size in all individuals. The fact that the difference in size between the polymorphic fragments was apparently very similar with different enzymatic cleavages led to the conclusion that this polymorphism was due to the presence or absence of a DNA sequence (designated hereafter insertion) within the ACE gene.
SUMMARY OF THE INVENTION
The invention follows from the location of the insertion in intron 16 (SEQ ID NO:3) of the gene, i.e. between exon 16 (SEQ ID NO:2)and exon 17 (SEQ ID NO:4) after the nucleotide structure of this intron had itself been determined. The sequencing of the intron, and more particularly of the regions flanking the insertion and the insertion itself has enabled the inventors to determine the nature of the insertion. It is a repetitive sequence of the Alu type.
Hence, the subject of the invention is products, in particular nucleotide fragments deriving from this observation, more particularly primers which can be used in gene amplification procedure, for example amplification by PCR ("polymerase chain reaction") type.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows to the general organization of intron 16 in alleles derived from subjects who possess the insertion (II) and from those who lack it (DD), respectively.
FIGS. 2 (a), 2 (b) and 2 (c) present the sequences of intron 16 and exons (exons 16 and 17 SEQ ID NO:2 and SEQ ID NO:4, respectively) which surround and flank intron 16 (SEQ ID NO:3) in the ACE gene. Only the nucleotides (nt) of intron 16 (SEQ ID:8) are numbered. FIG. 2a also includes intron 15 (SEQ ID NO:1). FIG. 2b also includes intron 17 (SEQ ID NO:5). FIG. 2c includes exon 18 (SEQ ID NO:6) and intron 18 (SEQ ID NO:7).
The sequence which is present or absent depending on the individual comprises the nucleotides 1451 to 1738 (SEQ ID NO:8).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates more particularly to any DNA fragment comprising at least 8 nucleotides and characterized by a sequence contained in the DNA of intron 16 (nt 1-1856 shown in FIG. 1) (SEQ ID NO:3) and, consequently, having a maximum of 1856 nucleotides. However, it relates more particularly to any one of the following fragments. nt. 1451-1738 (SEQ ID NO:8) (insertion); insertion. flanking the above-mentioned insertion. sequence nt. 1451-1738 (SEQ ID NO:8).
Advantageously, these various DNA fragments comprise from 15 to 40 nucleotides.
The invention relates more particularly to pairs of distinct fragments selected from those previously mentioned, which can be used to design primers which can be used in sequence amplification pr
REFERENCES:
Batzer et al., "Structure and Variability of Recently Inserted Alu Family Members", Nucleic Acids Research, 18, 6793-6798 (1990).
Lanzillo, "Chemiluminescent Nucleic Acid Detection with Digoxigenin-Labeled Probes: A Model System With Probes for Angiotensin Converting Enzyme Which Detect Less Than One Attomole of Target NDA", Analytical Biochemistry, 194, 45-53 (1991).
Rigat et al., "An Insertion/Deletion Polymorphism in the Angiotensin I-Converting Enzyme Gene Accounting for Half the Variance of Serum Enzyme Levels", Journal of Clinical Investigation, 86, 1343-1348 (1990).
Corvol Pierre
Hubert Christine
Soubrier Florent
Institut National de la Sante et de la Recherche Medicale
Jones W. Gary
Rees Dianne
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