EP1169478A2 - Procede d'identification d'organismes par analyse genetique comparative, amorceur et sondes d'hybridation utilises pour la mise en oeuvre de ce procede - Google Patents

Procede d'identification d'organismes par analyse genetique comparative, amorceur et sondes d'hybridation utilises pour la mise en oeuvre de ce procede

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Publication number
EP1169478A2
EP1169478A2 EP00918802A EP00918802A EP1169478A2 EP 1169478 A2 EP1169478 A2 EP 1169478A2 EP 00918802 A EP00918802 A EP 00918802A EP 00918802 A EP00918802 A EP 00918802A EP 1169478 A2 EP1169478 A2 EP 1169478A2
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Prior art keywords
species
gene
dna
pten
sequences
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EP00918802A
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German (de)
English (en)
Inventor
Hans Konrad Schackert
Matthias Hahn
Olga Niki Koufaki
Heike GÖRGENS
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Gorgens Heike
Schackert Hans Konrad
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Gorgens Heike
Schackert Hans Konrad
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Publication of EP1169478A2 publication Critical patent/EP1169478A2/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • 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

  • the invention relates to a method for the genetic analysis of organisms of various animal and / or plant species by examining coding and non-coding areas of highly conserved genes or pseudogenes and their homologs in various animal and plant species.
  • the known methods for the simple, fast and precise determination of gene sequences for the determination of kinship relationships and for the identification of organisms are based on the use of oligonucleotides which are specific for a species. These oligonucleotides are required in order to provide sufficient genetic material for the subsequent sequencing reaction by means of a polymerase chain reaction or other methods.
  • An o-nucleotide is usually a short synthetic produced molecule that attaches to a specific gene segment and has a specific sequence complementary to this strand.
  • the invention is therefore based on the object of a method for genetic engineering
  • Time expenditure provides reliable results, is also suitable for large series examinations and routine tests, and can also be carried out automatically if necessary. Furthermore, the object of the invention is to develop means for carrying out this method.
  • the invention relates to a method by which the gene sequence of a
  • Primer-serving oligonucleotides for the amplification of the DNA have to bind to regions of the genome that are highly conserved in order to ensure amplification of genetic material by means of an identical pair of oligonucleotides in all or the greatest possible number of different organisms.
  • Oligonucleotides must span an area that has a high sequence diversity between different species in order to enable differentiation.
  • the area that is spanned by the oligonucleotides used as primers should be as small as possible in order to maximize the yield of amplificates and to ensure that DNA degraded in the starting material!
  • this object is achieved by providing a method for
  • oligonucleotide pair Binds oligonucleotide pair to DNA sequences that are highly conserved between different species, thus allowing the amplification of a gene segment that is identical for all species.
  • the oligonucleotides include or several gene regions that have the greatest possible sequence differences between different species.
  • the determination of the gene sequence of this highly polymorphic gene region in a subsequent reaction step allows the gene sequence to be assigned to a specific species. Genotyping is carried out by sequencing or by other methods that are suitable for the detection of sequence variants.
  • PCR polymerase chain reaction
  • OLA oligonucleotide ligation assays
  • DNA isolation DNA is isolated and purified from blood samples, tissues, hair, foods and samples which contain DNA.
  • Polymerase chain reaction The polymerase chain reaction serves to amplify DNA for the subsequent sequencing reaction.
  • one or more pairs of oligonucleotides attach to the DNA to be analyzed (template DNA) of genes that are highly conserved between organisms of different species.
  • One of the molecules of an oligonucleotide pair is complementary to one of the two template DNA strands on the 5 ' or 3 ' flank of a DNA sequence.
  • the addition takes place in such an orientation that the polymer products obtained in an oligonucleotide-controlled polymerase chain reaction with in each case one of the two oligonucleotides, after denaturation, can serve as a template for the attachment of the other oligonucleotide.
  • the pair of oligonucleotides flank the area to be duplicated.
  • These oligonucleotides are extended in accordance with the nucleotide sequence of the matrix strand by means of a polymerase and after addition of nucleotide building blocks.
  • the addition, extension and denaturation take place at different temperatures and are usually carried out 20 to 35 times in succession, so that the area spanned by the oligonucleotides is multiplied exponentially.
  • c) Agarose gel electrophoresis In an agarose gel, the DNA fragments are separated from the oligonucleotides under a tension field and the band specific for the PCR product is cut out with a scalpel and purified.
  • d) Sequencing reaction Another polymerase is added, as well as all nucleotide building blocks and special nucleotide building blocks that lead to chain termination in the extension reaction. This creates DNA fragments that differ in length by one nucleotide each. Areas within the gene are sequenced which are polymorphic in their gene sequence between the different species. In this way, the DNA sequence characteristic of a species can be determined and assigned to a species.
  • Oligonucleotide pairs which attach to coding or non-coding regions of highly conserved genes or pseudogens and their homologs, that is to say to genes or pseudogens and their homologs which have little or no sequence differences between the individual species, are preferably used in the method for amplification according to the invention it is ensured that in the gene segment to be analyzed in each case an amplificate can be formed in a wide variety of species by means of a pair of primers.
  • regions of the gene or pseudogen and their homologs are preferably analyzed which differ in their gene sequence between organisms of different species. These can be sequences of coding or non-coding DNA.
  • oligonucleotides of different lengths are used in a reaction mixture (multiplex-PCR), all of which partially match an initial sequence.
  • the sense or antisense oligonucleotides differ with respect to the nucleotide sequence in that some oligonucleotides differ in length in the 3 ' region by one or more nucleotides.
  • the use of several oligonucleotides with different lengths is intended to ensure that an attachment of the oligonucleotides to the template DNA is possible even if the template DNA differs in the 3 ' region from some of the oligonucleotides used.
  • the agreement at the 3 ' region of the oligonucleotides with the template DNA is essential for the specific amplification and should therefore be as precise as possible in this region.
  • multiple pairs of oligonucleotides are used in a multiplex PCR which are identical in terms of their length, but differ in one or more positions at the 3 ' end of the oligonucleotides with respect to the nucleotide sequence.
  • the use of a reaction mixture of several oligonucleotides which differ in the base sequence at the 3 ' end of the oligonucleotide is intended to ensure that one
  • HETZBLATT (RULE 26) an amplificate can be formed in a wide variety of species by means of a pair of primers.
  • regions of the gene or pseudogen and their homologs are preferably analyzed which differ in their gene sequence between organisms of different species. These can be sequences of coding or non-coding DNA.
  • oligonucleotides of different lengths are used in a reaction mixture (multiplex-PCR), all of which partially match an initial sequence.
  • the sense or antisense oligonucleotides differ with respect to the nucleotide sequence in that some oligonucleotides differ in length in the 3 ' region by one or more nucleotides.
  • the use of several oligonucleotides with different lengths is intended to ensure that an attachment of the oligonucleotides to the template DNA is possible even if the template DNA differs in the 3 ' region from some of the oligonucleotides used.
  • the agreement at the 3 ' region of the oligonucleotides with the template DNA is essential for the specific amplification and should therefore be as precise as possible in this region.
  • multiple pairs of oligonucleotides are used in a multiplex PCR which are identical in terms of their length, but differ in one or more positions at the 3 ' end of the oligonucleotides with respect to the nucleotide sequence.
  • the use of a reaction mixture of several oligonucleotides which differ in the base sequence at the 3 ' end of the oligonucleotide is intended to ensure that one
  • oligonucleotides are also attached to the template DNA if the template DNA sequence at the 3 ′ binding site of the oligonucleotide differs from the oligonucleotide that is usually used. For this purpose, a mixture of different oligonucleotides, which have all conceivable nucleotide sequences at the 3 ' end, is provided for the amplification.
  • oligonucleotide sequences in the method according to the invention, care is preferably taken to ensure that as many of the oligonucleotides at the 3 ' end as possible match the first nucleotide of the codon which codes for the highly conserved amino acid. From theoretical considerations and also from observations it follows that the second nucleotide within a codon has a greater degree of correspondence between organisms of different species than the first or third nucleotide of the codon.
  • the nucleotide at the 3 ' end of the oligonucleotide is also exactly complementary to the opposite nucleotide of the template DNA strand - that is, an A is a T or a G is a C - the sequence of the oligonucleotides used is chosen such that the last nucleotide at the 3 ' end of the oligonucleotide is attached to the most conserved nucleotide of the codon which codes for an amino acid.
  • a single pair of oligonucleotides is used for different sections of the gene, the pseudogenes and its homologues, which pairs with the highly conserved tumor suppressor gene PTEN / MMAC1, its pseudogen and their homologues different species, namely in the region of the gene that shows a high degree of agreement between the species [Steck et al., 1997].
  • This area concerns the entire coding sequence of the gene, pseudogen and their homologs as well as exon-intron transitions and 5 'and 3' untranslated regions of the gene, the pseudogen and their homologs.
  • the regions amplified with the methods described above contain sequence sections which have more or less large sequence differences between the individual species. This is particularly true for intron regions and especially for intron 4.
  • the pair of oligonucleotides used for the amplification can bind to exons 4 and 5, since the PTEN / MMAC1 gene, the pseudogen and their homologs between the species in exon 4 and exon 5 do not or shows very little difference.
  • Comparative analyzes can be used to determine whether different samples belong to an identical species based on the agreement of intron sequences. Furthermore, the comparative analysis of an intron region of the PTEN / MMAC1 gene and its homologs comprising a few to several hundred bases can determine the degree of kinship of different species based on the similarity of the sequences. In general, longer intron sections have to be examined for the differentiation of closely related species than for distantly related species. This procedure described for exon 4/5 and intron 4 can also be applied to all other introns that are enclosed by exons. It also applies to pure exon regions, pseudogenes and the 5'- and 3'-untranslated region, as well as their homologues, in which case the sequence differences between the individual species are smaller than in the intron regions.
  • pseudogenes and their homologs are examined in various organisms and to determine the species-specific gene sequence used. These pseudogenes and their homologs offer the advantage that they are also highly conserved in their nucleotide sequence and allow amplification in certain species. However, the sequence differs in some areas of the pseudogen and its homologues between different species, so that pseudogenes and their homologues can be used for the species-specific characterization of organisms. Since the intron regions are missing, these, like pure exon regions, and the 5'- and 3'-untranslated regions, due to their small size, are particularly suitable for the analysis of DNA which is degraded due to environmental influences.
  • An advantageous implementation of the method uses the 9 base pair deletion identified by the inventors in a PCR product (see FIG. 3 and example 2) which corresponds to a gene sequence variant of PTEN / MMAC1 and was amplified by DNA from pig cells.
  • This 9 base pair deletion is typically found in domestic pigs and all examined wild boars and otherwise not in any of the examined species.
  • this difference in length is used to detect pork in food.
  • This length variant is genotyped by sequencing or by other methods which are suitable for the detection of this deletion. This includes PCR-supported genotyping methods such as PCR using species-specific oligonucleotides.
  • Hybridization techniques such as the light cycler technology but also other genotyping methods using restriction enzymes, as well as in principle any currently and future available method for variant detection including the chip technology in all its technological versions.
  • deletions and insertions such as those found in various species in intron 4, exon 8 and in the 5'-untranslated region, can be used appropriately for species identification (see Appendix: “Compilation of the species sequences”).
  • the method according to the invention was carried out using DNA from various species as a model system and the gene sequence in the region of two sections in intron 4 of the PTEN / MMAC1 gene and its homologs was determined. It was possible to amplify all species with only one pair of oligonucleotides in the sequencing reaction 10 using only one oligonucleotide and in the subsequent analytical polyacrylamide gel electrophoresis, it was evident that all the examined species differ in the nucleotide sequence in the intron region (see Appendix: "Compilation of the species sequences").
  • the method is due to the conservatism of the PTEN / MMAC1 tumor suppressor and Its pseudogen and its homologues in other species are also suitable for successfully amplifying the corresponding gene sequences in lower organisms and possibly plants using an oligonucleotide pair.
  • the method according to the invention is therefore suitable for determining the identity and relationship of different organisms.
  • a database has been established on which can be used in the identification of different species and humans and includes PCR primers, sequencing primers and hybridization probes, as well as sequences of the coding and non-coding regions including ch selected highly variant intron regions, exon regions, the 5'-untranslated region of the gene and its homologues as well as the pseudogen and its homologues from various vertebrates (see Appendix: "Compilation of the species sequences").
  • the method according to the invention is suitable for quickly, easily and reliably reproducibly determining the degree of relatedness in certain species which are clearly classified (phyiogenetic analyzes).
  • the identity of tissue samples, blood samples and food and all samples which contain DNA and are of unknown origin can be determined by comparison with gene sequences which can be clearly assigned to a species.
  • the method is therefore also suitable for applications in forensic medicine. Since DNA is used as the starting material in a preferred embodiment of the method according to the invention, organic samples (eg blood, saliva, tissue residues) can be clearly assigned to human or animal origin based on their gene sequence. Because of the possibility of comparing the DNA sequence obtained with an already established database with DNA sequences of known species, statements regarding the species can be made. If the gene sequence is unknown, statements can be made on the basis of sequence similarities as to what degree of relationship the species to be examined has with a comparative DNA.
  • An advantageous embodiment of the invention uses hybridization probes to distinguish the DNA of different species from one another.
  • the method uses different methods to differentiate the species based on the LightCycler analysis system (Röche Molecular Biochemicals) using hybridization probes (patents WO 97/46714, WO 97/39008 )
  • the LightCycler analysis system With the LightCycler analysis system, the amplification, detection and specific analysis of DNA of different species and their differentiation is possible in a very short time and with relatively little effort.
  • the LightCycler is a micro-volume fluorometer with a thermocycler that enables fast thermocycling with real-time fluorescence observation combined during the PCR (Wittwer et al, 1997a) With this technology, the time for the amplification and detection of nucleic acids is reduced from approx. 5 hours to approx. 30 minutes.
  • the synthesis can be based on the fluorescence Resonance energy transfers (FRET) can be observed via two adjacent hybridization probes labeled with fluorescent dyes.
  • FRET fluorescence Resonance energy transfers
  • One of the probes at the 3 ' end is marked with a donor fluorophore (usually fluorescence) and the neighboring probe at the 5' end with an acceptor fluorophore of the FRET, the donor dye is applied via an external light source excites and emits light which is absorbed by the acceptor fluorophore which in turn emits light of a different wavelength which is specifically measured
  • This FRET can only be carried out if the two probes side by side within the amplification pair at a distance of approx.
  • the LightCycler can be used to carry out melting point analyzes.
  • the melting point analysis is based on the fact that two complementary DNA strands separate at a characteristic temperature, the melting temperature into two single strands.This melting temperature depends on the base composition in that GC-rich sequences have a higher melting point than sequences with predominantly AT bases. If you carry out a melting point analysis by choosing more or less complementary hybridization probes under certain
  • a melting curve in the form of the fluorescence intensity as a function of the temperature can be created for each sample. Comparing the melting peaks created by the first negative derivative of the fluorescence to temperature (-dF / dt vs T) enables the verification and differentiation of different DNAs. Fragments with a lower or higher
  • oligonucleotide pairs for the polymerase chain reaction have been found which attach to the highly conserved tumor suppressor gene PTEN / MMAC1, its pseudogen and their homologues in areas which show a great match between the species. These are mostly exon regions or untranslated regions of the 3 'and 5' end of the gene, its pseudogen and their homologues.
  • Examples 4, 5 and 6 use a 9-base pair deletion of the pig homolog of the PTEN / MMAC " / pseudogen and numerous other sequence variants of the gene, the pseudogen and their homologs in different species to use different hybrid profiles with the help of specific hybridization probes to produce different species that clearly differentiate the species from each other, and this preferred range of Examples 4-6 only concerns exon 5 of the gene, pseudogen and their
  • the PCR primers used for the amplification of the corresponding gene section are:
  • Antisense primer Zoo44aRV 5 ' - ttgt ctc tgg tcc tta ctt c-3 '
  • Hybridization probes according to the invention were selected on the one hand with the target region of the 9-base pair deletion of the pig pseudogenes.
  • the A1 / A2 probe enables pig DNA to be distinguished from all other species.
  • Probes A1 / A2 specific for PTE / V pseudogene pig:
  • Probes B1 / B2 specific for PTE / V pseudogene human:
  • Probes C1 / C2 specific for PTE / V homologue pig: C1: 5 ' - tgc ata ttt gtt aca tcg ggg taa att - fluorescein - 3 ⁇ C2: corresponds to probe B2
  • the positions of the probes A1, A2, B1, B2, C1 and C2 in exon 5 are shown in FIG. 4.
  • the use of these three pairs of probes individually and the use of different combinations of the probes with one another (1 donor dye + 1 acceptor dye) results in a characteristic panel of different melting points for each individual species, which enables a clear differentiation of the species from one another (see FIGS. 5 and 7)
  • the use of these hybridization probe combinations allows investigations in reaction mixtures of two and more different species
  • a parallel analysis / detection of the fluorescence from two different wavelengths is possible in a multiplex reaction with two different pairs of probes in such a way that the respective donor probes are marked differently.
  • the sequence of the acceptor probes can be identical or different.After melting point analysis is obtained for each of the two wavelengths has a melting point specific to the corresponding probe and the target sequence it covers
  • the following general multiplex reaction approaches are possible, which are characterized in that at least one pair of hybridization probes is used and at least one gene section is amplified, different pairs of hybridization probe hybridize to different gene sections, and the melting points are determined by the different combinations and put together for each species to form a panel or used for identification
  • probes and / or P ⁇ mer can be combined according to the multiplex principle with the aim of species differentiation
  • all of the methods described, as well as all currently and in the future conceivable methods for analyzing the DNA sequence variants can be applied to both the sense and the antisense strand.
  • This principle of equivalence also applies to all PCR, sequencing primers and hybridization probes which are described in this patent application and the gene sequences described for the individual species.
  • Oligonucleotide 1 5 ' - cga cgt tgt aaa acg acg gcc agt tgt gct gag aga cat tat gac - 3 '
  • Oligonucleotide 2 5 ' - cag gaa aca gct atg act tgt ctc tgg tcc tta ctt c- 3 '
  • the oligonucleotides for the polymerase chain reaction were constructed so that they each have (underlined) a sequence at their 5 ' end that is complementary to the two oligonucleotides used in the sequencing reaction. This ensures that the specific parts of the oligonucleotide bind specifically to the template DNA (not underlined), but that other oligonucleotides can be used for the sequencing than for the polymerase chain reaction, which generally increases the quality of the sequencing reaction.
  • the specific proportion of the oligonucleotides binds to exon 4 and exon 5 of the PTEN / MMAC1 tumor suppressor gene and its homologues. As part of a routine procedure, 3 ml of the resulting amount were removed by a veterinarian
  • Kits (QIAGen) DNA was isolated from elephant blood and human blood according to the manufacturer's protocols and used until -
  • a polymerase chain reaction (PCR) was carried out to generate sufficient amounts of DNA for the sequencing reaction. There were 3
  • reaction vessel 1 Amounts of substrates and units of polymerase used: reaction vessel 1:
  • Reaction tube 3 no DNA, instead 1 ⁇ l distilled water. (Negative control). all
  • a 0.8% agarose gel was prepared with the addition of ethidium bromide and 1 x TAE buffer was added. Each 13 ⁇ l PCR product with the addition of 2 ⁇ l of dye was pipetted into prepared sample pockets in the gel matrix and a voltage of 100 V was applied. After 20 to 30 minutes, the electrophoresis was stopped
  • oligonucleotides with the sequence 5 ' - Cy-5 - cag gaa aca gct atg ac -
  • reaction vessels (A, C, G, T) were provided for each PCR product and the following volumes and concentrations were selected: 3 ⁇ l PCR product for A, C, G, T; 1 ⁇ l each of A, C, G, T, reagent (Amersham Pharmacia) consisting of Tris-HCL (pH 9.5),
  • the gel matrix was composed of: 16.8 g urea (Gibco BRL), 5.2 ml
  • Reaction tube A, C, G, T, 5 ⁇ l formamide loading dye were added and pipetted into the prepared sample pockets.
  • the following electrophoresis conditions were selected: 1000 V, 40 mA, 40 W.
  • FIG. 2 shows the nucleotide sequence differences determined.
  • oligonucleotides labeled with Cy-5 were sequenced with the sequence 5 ' - Cy-5-cag gaa aca gct atg ac -3 ' .
  • the comparative analysis of pig liver from the butcher's shop with DNA of a pig already sequenced in this area showed complete agreement.
  • the control DNA (bovine salami) has a clearly different sequence from the pig DNA (see FIG. 3).
  • Hybridization probe pairs are shown. The comparative analysis was carried out by determining the melting points of the hybridization probe pairs C1 + B2, A1 + B2, A1 + A2 and C1 + A2 in pig DNA and human DNA were. The probes were selected and synthesized accordingly (Tib Mol
  • the prerequisite for probe hybridization is amplification of the target area with a suitable pair of primers.
  • the following pair of primers was selected and synthesized:
  • Antisense primer Zoo44aRV 5 * - ttgt ctc tgg tcc tta ctt c-3 ' (AmershamPharmacia)
  • Both primers bind to regions of exon 5 of the PTEN / MMAC1 gene, the pseudogen and their homologues, which includes the target region of approximately 172 base pairs.
  • the DNA was isolated from pork and human blood using the Quiagen Kit (QIAGen) according to the manufacturer's protocols and stored at -20 ° until use.
  • the real-time PCR with hybridization probes and subsequent melting point analysis was carried out as follows: 3 rows, each with 4 sample batches (glass capillary cuvettes (Röche), were provided in a cooled pipetting block (Röche).
  • row 1 2 ⁇ l (50ng) pigs were added to each batch -DNA submitted, 2 ⁇ l (50ng) human DNA in each batch of the 2nd row and 2 ⁇ l distilled water for the negative controls in the 3rd row
  • the hybridization probes were used for the corresponding batches in the following combinations: 1st batch of each row: A1 , 1 ⁇ l (0, 1 ⁇ M) and A2, 2 ⁇ l (0.2 ⁇ M); 2nd batch of each row: C1, 1 ⁇ l (0, 1 ⁇ M) and B2, 2 ⁇ l (0.2 ⁇ M), 3 batches of each row A1, 1 ⁇ l (0.1 ⁇ M) and B2, 2 ⁇ l (0.2 ⁇ M),
  • Oligonucleotides PTEN se and Zoo44aRV each 2 ⁇ l (10 ⁇ M), MgCL 2 (Röche Molecular
  • the tempering rate is from denaturation to storage at 20 ° C /
  • the melting points of the pig DNA could be compared with those of the human DNA for the different probe combinations. DNA, 2 melting points were recorded for each pair of probes, one for the pseudogen and one for the gene. All melting points of the pig DNA were different from those of the human DNA (FIG. 5)
  • the comparative analysis was carried out by determining the melting points of a hybridization probe pair, the sequences of which are specific for the pseudogen of the pig in the region of the 9 base pair deletion (A1 / A2) and which were already used in Example 4
  • the DNA used was obtained from the bleeds of the species mentioned using QIAGen K ⁇ t (QIAGen) and purified.
  • QIAGen QIAGen K ⁇ t
  • a sample batch with 2 ⁇ l (50 ng) of the corresponding DNA is used for each species and a sample batch for a negative control presented with 2 ⁇ l distilled water 1 ⁇ l (1 ⁇ M) of probe A1 and 2 ⁇ l (0.2 ⁇ M) of probe A2 were pipetted into each sample batch.
  • the procedure was followed for pipetting further reagents in their volumes and concentrations and for further reaction steps on the LightCycler chosen under example 1
  • the comparative analysis was carried out by determining the melting points from various combinations of the hybridization probes from Example 4 in the combinations C1 + B2, A1 + B2, A1 + A2, C1 + A2, B1 + B2 and B1 + A2 and one for each animal species Panel were put together.
  • the DNA was obtained from the bleeds of the species mentioned using the QIAGen Kit (QIAGen) and purified.
  • the primers listed in Example 4 were used for the amplification.
  • the procedure, expanded by the additional probe combinations and larger number of species, was chosen under Example 4.
  • sequences of the examined species for exon 1-9 and the 5 ' untranslated region of the PTEN / MMAC1 gene, the pseudogen and their homologs are listed in the appendix under "Compilation of the species sequences".
  • These examples of different probe combination panels can be expanded and used by any number of probes and primers for the described gene PTEN / MMAC1, the pseudogen and its homologues, but also for all other genes.
  • a linear increase in the number and combination of probes in the entire genome results in an exponential increase in the possibility of differentiation of different species from one another.
  • sequence-specific probes and several and / or any number of combinations of probes a species can be distinguished from all other species by its very specific melting point panel
  • HPA-1 human platelet antigenl
  • Figure 1 Schematic representation of the method for
  • Figure 2 Comparison of the determined sequences of humans and African elephants according to Example 1
  • Figure 3 DNA sequence comparison of a pig with commercially available pig liver and bovine salami according to Example 2. The differences in the nucleotide sequences are shown. The 9-base pair large deletion (nucleotides 216-224) in the pig / pig liver is striking.
  • Figure 5 Example 4 - melting point panel of pig and human

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé, un amorceur et des sondes d'hybridation utilisés pour l'identification d'organismes par une analyse génétique par comparaison. Ce procédé consiste à amplifier des zones codantes, non codantes et/ou des zones d'importance fonctionnelle de gènes à haute conservation, de pseudogènes ou d'homologues à l'aide de la PCR puis à les génotyper et à les analyser. La comparaison de zones codantes et de zones non codantes de gènes à haute conservation, de pseudogènes ou d'homologues garantit qu'une seule paire d'oligonucléotides se lie à des séquences d'ADN à haute conservation de différentes espèces. Cela permet l'amplification d'une section génique identique pour toutes les espèces. Les oligonucléotides comportent au moins une zone génique présentant, si possible, de grandes différences de séquences entre les différentes espèces. La détermination de la séquence génique de cette zone hautement polymorphe lors d'une réaction consécutive permet d'affecter la séquence génique à une espèce spécifique. Dans des modes de réalisation préférés, on a trouvé des paires de nucléotides qui permettent l'amplification du gène à haute conservation destiné à la de suppression tumorale PTEN/NMAC1, de son pseudogène et de son homologue.
EP00918802A 1999-03-16 2000-03-16 Procede d'identification d'organismes par analyse genetique comparative, amorceur et sondes d'hybridation utilises pour la mise en oeuvre de ce procede Withdrawn EP1169478A2 (fr)

Applications Claiming Priority (5)

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DE19911656 1999-03-16
DE19911656 1999-03-16
DE19964112 1999-12-31
DE19964112 1999-12-31
PCT/EP2000/002330 WO2000055361A2 (fr) 1999-03-16 2000-03-16 Procede d'identification d'organismes par analyse genetique comparative, amorceur et sondes d'hybridation utilises pour la mise en oeuvre de ce procede

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EP1169478A2 true EP1169478A2 (fr) 2002-01-09

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EP00918802A Withdrawn EP1169478A2 (fr) 1999-03-16 2000-03-16 Procede d'identification d'organismes par analyse genetique comparative, amorceur et sondes d'hybridation utilises pour la mise en oeuvre de ce procede

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EP (1) EP1169478A2 (fr)
JP (1) JP2002538835A (fr)
AU (1) AU3962100A (fr)
DE (1) DE10014575A1 (fr)
WO (1) WO2000055361A2 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10111420A1 (de) * 2001-03-09 2002-09-12 Gnothis Holding Sa Ecublens Bestimmung von Analyten durch Fluoreszenz-Korrelationsspektroskopie
US7785776B2 (en) * 2002-05-13 2010-08-31 Idaho Technology, Inc. Genotyping by amplicon melting curve analysis
DE10313791A1 (de) * 2003-03-20 2004-10-07 Bange, Franz-Christoph, Dr. Spezifischer Nachweis von Mykobakterien des M. tuberculosis-Komplex
FR2873716B1 (fr) * 2004-07-30 2008-07-04 Univ Claude Bernard Lyon Procede permettant de detecter et d'identifier simultanement differentes especes animales ou vegetales, dans un echantillon de matiere organique
DE102014221734A1 (de) * 2014-10-24 2016-04-28 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Messvorrichtung und System zur Schmelzkurvenanalyse eines DNA Microarrays, sowie Verwendung eines Fluoreszenzdetektorarrays zur Analyse

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5614361A (en) * 1981-09-25 1997-03-25 Webster, Jr.; John A. Method for identifying and characterizing organisms
CA2026264A1 (fr) * 1990-09-26 1992-03-27 William Scott Davidson Test pour determiner l'identite de l'espece et(ou) de la population d'un organisme par l'analyse directe de la sequence des nucleotides de segments definis du genome
US5849492A (en) * 1994-02-28 1998-12-15 Phylogenetix Laboratories, Inc. Method for rapid identification of prokaryotic and eukaryotic organisms
DE19629166C2 (de) * 1995-08-22 1997-09-18 Ztb Zentrum Technologietransfe Analytisches Verfahren zur Feststellung der Abstammung verschiedener Materialien biologischer Herkunft unter Verwendung mindestens eines Primers
EP0892808B1 (fr) * 1996-04-12 2008-05-14 PHRI Properties, Inc. Sondes, trousses et dosages de detection

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BERNARD P.S. ET AL: "Homogenous Muliplex Genotyping of Hemochromatosis Mutations with Fluorescent Hybridization Probes", AMERICAN JOURNAL OF PATHOLOGY, vol. 153, no. 4, 1998, pages 1055 - 1061 *
See also references of WO0055361A3 *

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AU3962100A (en) 2000-10-04
JP2002538835A (ja) 2002-11-19
DE10014575A1 (de) 2000-11-30
WO2000055361A2 (fr) 2000-09-21
WO2000055361A3 (fr) 2001-10-18

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