EP1002133A2 - Utilisation d'amorces dans l'analyse d'empreintes - Google Patents

Utilisation d'amorces dans l'analyse d'empreintes

Info

Publication number
EP1002133A2
EP1002133A2 EP98943846A EP98943846A EP1002133A2 EP 1002133 A2 EP1002133 A2 EP 1002133A2 EP 98943846 A EP98943846 A EP 98943846A EP 98943846 A EP98943846 A EP 98943846A EP 1002133 A2 EP1002133 A2 EP 1002133A2
Authority
EP
European Patent Office
Prior art keywords
primer
primers
istr
use according
pair
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP98943846A
Other languages
German (de)
English (en)
Inventor
Wolfgang Rohde
Dieter Becker
Francesco Salamini
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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 Max Planck Gesellschaft zur Foerderung der Wissenschaften eV filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority to EP98943846A priority Critical patent/EP1002133A2/fr
Publication of EP1002133A2 publication Critical patent/EP1002133A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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 the use of primers or pairs of primers for DNA fingerprint analysis, fingerprints being obtainable with the primers or pairs of primers both from humans and from animals, and from plants and from microorganisms.
  • the invention further relates to primers or pairs of primers for the use mentioned above and to kits which contain the primers or pairs of primers.
  • retrotransposons such as the Drosophila copia elements and copia-like elements in other species of the animal and plant kingdom, are usually contained as multiple copies in genomes. Repetitive genome sequences of this type have been used for the genetic analysis of these plant species using the example of copia-like elements in pisum (pea) (Lee et al., Plant Mol. Biol. 15: 707-722, 1990).
  • This method which the authors call OFLP, is based on a copia-specific primer and as a second primer for the PCR amplification of a sequence from the pea genome flanking these retrotransposons.
  • primers that are closer to the following hybridize marked areas from the copia-like element from the coconut (Cocos nucifera L.), and enable fingerprint analysis there, also with many other species from the animal and plant kingdom, including yeast, as well as with humans and even microorganisms, with success can be used. This finding permits the universal applicability of the primers mentioned for fingerprint analysis in the entire animal and plant kingdom as well as in humans and in microorganisms.
  • the invention thus relates to the use of a primer or pair of primers for DNA fingerprint analysis, which is characterized in that with the primer or the pair of primers, a fingerprint is obtainable both from humans and from animals, and from plants and from microorganisms , and wherein the primer or pair of primers hybridizes to DNA encoding the endonuclease, reverse transcriptase or RNAse H of a copia or copia-like element, in particular from coconut (Cocos nucifera L.).
  • the primer / the primer pair hybridizes with organisms from at least one species from the aforementioned taxonomic groups.
  • primers of opposite orientation which only have to meet the condition that they hybridize to the aforementioned DNAs, and with the use of a single primer which, owing to the repetition of the copia- or copia-like element, however in 5 '- »373' -> 5 'orientation of two adjacent elements and not as shown in FIG. 2B, in ⁇ ' ⁇ S '/ ⁇ ' ⁇ S 'orientation, likewise the highly polymorphic Fingerphnts provides.
  • the definition of primers chosen above naturally includes the fact that these also hybridize to DNAs of other organisms, provided that these contain DNA sequences which correspond to DNA sequences from the above-mentioned copia or copia-like element.
  • the conditions under which the primers are hybridized and subsequently amplified can be derived by the person skilled in the art from the prior art and the examples below without inventive effort. Suitable conditions for the hybridization of the primers and / or the subsequent amplification can be found, for example, in the textbook Sambrook et al., "Molecular Cloning, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1989 (hereby incorporated by reference) or also following examples.
  • the overlap region comprises at least 1 nucleotide, preferably at least 5 nucleotides and particularly preferably at least 10 nucleotides.
  • the length of the primers used in this invention is preferably 15 to 30 nucleotides. However, the invention can also be carried out with shorter or longer primers.
  • This subfamily of copia-like sequences in the coconut genome is therefore a sequence which is repeated in tandem and which has homology to the endonuclease and reverse transcriptase / RNAseH region of a copia or copia-like element (see FIG. 1B).
  • the observed sequence differences in the elements of this subfamily - in contrast to the experiments described above for the Ecorep sequences - could now be used with the above-described suitable PCR primer for genetic analysis in coconut.
  • This method for genome analysis in coconut was called ISTR (inverse sequence-tagged repeat) analysis.
  • the primer or the primer pair comprises a fingerprint with DNAs from the entire microorganism, animal and plant kingdom (a) the animal kingdom with all sub-kingdoms, preferably the metazoa, therein contain the sub-strains of the vertebrates, preferably the class of the mammals, in particular contain the family of the hominids and the family of the Bovidae, therein the species Bovis taurus and Ovis aries, as well as all breeds and varieties that can be derived from the corresponding species;
  • the plant kingdom with all subranges preferably that of the Mycobionta and Cormobionta, in the latter preferably the department of spermatophytes, preferably the class of the Monocotyledonae with their families of the Areaceae and their representatives of the species Cocos nucifera or the family of the Poaceae with their representatives of the species Hordeum vulgäre and Zea mays, more particularly preferred the class of the Dicotyledonae with their families, eg the Solaneceae and its representative of the species Solanum tuberosum, Nicotiana tabacum, Petunia hybrida, or e.g. the family of the Brassicaceae with their representative of the species Brassica napus or the family of the Chenopodiaceae with their representative of Beta vulgaris and all varieties and varieties which can be derived from the corresponding species;
  • microorganisms comprising prokaryotic microorganisms, preferably Gram-positive bacteria such as e.g. Lactic acid bacteria, Sarcina and Coryneform bacteria and Gram-negative bacteria such as Neisseria and enterobacteria, and eukaryotic microorganisms comprising fungi, preferably phycomycetes such as e.g. Phytophthora and Ascomycetes such as Yeast is available.
  • Gram-positive bacteria such as e.g. Lactic acid bacteria, Sarcina and Coryneform bacteria and Gram-negative bacteria such as Neisseria and enterobacteria
  • eukaryotic microorganisms comprising fungi, preferably phycomycetes such as e.g. Phytophthora and Ascomycetes such as Yeast is available.
  • fingerprints of comparable resolution and sensitivity with DIG-labeled PCR products are made visible directly in the gel without the transfer of the DNA fragments to membranes (Southern blot) which is generally carried out in a known manner.
  • This enables the creation of such fingerprints in the simplest way (separation of the PCR fragments in the sequence gel, direct detection in the gel, computer-assisted data analysis by direct scanning of the sequence gels) without the use of radioactivity.
  • a further preferred embodiment of the use according to the invention is thus characterized in that the DNAs to be analyzed are amplified with the primer or the primer pair by PCR and subsequently separated in size on a gel.
  • the gel is a sequence gel.
  • sequence gels The preparation of sequence gels is also known in the art and is described, for example, in Sambrook et al., "Molecular Cloning, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1989.
  • the use according to the invention is characterized in that a Southern blot is carried out as a further step and the DNAs transferred to the membrane are made visible by hybridization with a probe.
  • This embodiment is to be seen as an alternative to the two embodiments described above. Although it requires more effort and dealing with radioactivity, it is definitely suitable for laboratories that operate less complex laboratory equipment, e.g. do not have a scanner with a computer connected to it. Southern blots and hybridizations with a suitable probe are also known in the art and are described, for example, in Sambrook et al., Cited above.
  • the probe is the primer according to the invention or the primer pair according to the invention.
  • the primers are part of the amplified DNA, they can also be used to easily detect the bands on the membrane used for the Southern blot.
  • the primer or the primer pair bears a label.
  • the label is a non-radioactive label, in particular digoxigenin, biotin or a fluorescent dye, a dye or a radioactive label, in particular 32 P.
  • the labeling of the primers with digoxigenin and the staining after amplification of the DNA and gel electrophoresis separation Directly in the gel can be used by all laboratories or interested breeders using a small amount of equipment (PCR reaction, electrophoresis on sequence gels) and without radioactivity.
  • the data is preferably stored and processed by reading the colored and dried gel directly into a computer using a scanner.
  • it is possible to develop specific primers by isolating PCR products from the sequence gel, reamplification and sequencing, which give allele-specific amplification products.
  • the primer has one of the sequences given in Table 2.
  • primers are preferred examples of the primers used by the inventors in previous DNA fingerprint analyzes.
  • the primer has a sequence which overlaps with one of the sequences shown in Table 1 or 2.
  • the overlap region comprises at least 1 nucleotide, preferably at least 5 nucleotides and particularly preferably at least 10 nucleotides.
  • sequence of these primers which can be used in the use according to the invention can be determined by standard methods, for example by sequencing the sequences which are adjacent to the oligonucleotide sequences shown in Tables 1 and 2 in the copia- or copia-like elements.
  • overlapping sequences also includes sequences, one of which is completely encompassed by another.
  • the use according to the invention is characterized in that the fingerprint analysis for biodiversity studies, studies on genetic relationships, taxonomic studies, and in particular in forensic medicine, breeding, plant variety protection, in genebanking Management that uses population genetics and evolution studies.
  • the invention relates to primers for use according to the invention, which are characterized in that the primers have one of the sequences shown in Table 2 or a sequence which overlaps with one of the sequences shown in Table 1 or 2.
  • the invention further relates to kits which contain at least 1 primer and preferably at least 1 pair of primers which hybridize to the copia-like element of the coconut shown in FIG. 2b or which have been described above.
  • the primers preferably have the sequences shown in Tables 1 and / or 2 or sequences overlapping them.
  • the above primers can be packaged in containers, for example in vessels, optionally in buffers and / or solutions. If appropriate, one or more of the primers can be packaged in the same container.
  • the kits according to the invention can be used in a variety of ways. Exemplary areas of application such as breeding have been given above.
  • the invention also relates to the use of the primers described above for the production of the kits according to the invention.
  • the kits themselves are preferably produced using standard processes.
  • the invention relates to the use of primers which hybridize to the copia-like element of coconut shown in FIG. 2b, the primers preferably belonging to one of the groups defined in more detail above, for detecting recombination events at crossings, in particular in animal and Plant growing.
  • Fig. 1 Region of a copia-like element Bare-1 occurring in the barley genome (Fig. 1A, from Manninen and Schulman, Plant. Mol. Biol., 22: 829-846, 1993), which is repeated in tandem as copia-like Sequence (Rohde et al., J. Genet. & Breed., 49: 179-186, 1995) was found in the genome of the coconut (Cocos nucifera L.) (Fig. 1 B). (A) Schematic representation of the copia-like BARE-1 element made from barley.
  • ED endonuclease
  • RT reverse transcriptase
  • RH RNAse H
  • Fig. 2 Amplification of the "spacer region" between adjacent copia-like sequences in the coconut genome (A) and approximate position of primers previously used for ISTR analysis (B).
  • the individual primers are generally between 18 and 20 nucleotides long and were synthesized analogously to the sequence of the Ecorepl element (Rohde et al., J. Genet. & Breed., 49: 179-186, 1995).
  • the primers provided with "-" are complementary to the coding sequence of the copia element and can be combined with any primer of the "plus” series for ISTR analysis.
  • Fig. 3 ISTR analysis of populations using the example of coconut (from Rohde et al., J. Genet. & Breed., 49: 179-186, 1995).
  • Fig. 4 General application of ISTR primers in the plant area. DNA from various plant species was subjected to amplification with the primers ISTR5 / ISTR-2. The PCR products of the following plants were applied in the individual lanes: 1: tobacco, 2: barley, 3: potato, 4: maize, 5: antirrhinum, 6: Arabidopsis, 7: rapeseed, 8: craterostigma, 9: petunia , 10: parsley, 11: sisal, 12: milala palm, 13: borassus palm, 14: coconut palm, 15: sugar beet, 16: cuphea, 17: yeast.
  • Fig. 5 ISTR analysis of individual members of the Arecaceae family (Palmae). DNAs from 17 different palm species were amplified in a standard PCR reaction with the primers ISTR5 / ISTR-2 and separated on a 4% PAGE gel. The PCR products of the following plants are plotted in the individual lanes: 1: Hyphaene petersiana Mail .; 2: Bismarckia nobilis Hildebrandt & H. Wendl .; 3: Eugeissona utilis Becc; 4: Korthalsia echinometra Becc; 5: Mauritiella aculeata (H.B.
  • Fig. 6 ISTR analysis of barley varieties.
  • GSM marker (lower band of the triplet) that co-regulates with the male gender.
  • V father. The individual offspring of the different breeds are numbered.
  • Fig. 8 Analysis of three human families I, II and III with different primer pairs.
  • Figure 9 shows the DNA analysis of wine varieties.
  • ISTR fingerprint analysis DNA from 19 different wine types was subjected to a PCR reaction with the primer pair ISTR5 / ISTR-2.
  • the PCR products of the following plants were applied to the individual lanes:
  • the individual lanes are DNA fingerprints of the following genomic DNAs. 1: EAT (coconut); 2: PRD (coconut); 3: SRT (coconut); 4: human; 5: hamster; 6: rust fungus.
  • Lane 1 EAT (coconut); 2: PRD (coconut); 3: SRT (coconut); 4: human; 5: rape; 6: barley.
  • DNAs from various rust fungus isolates were amplified in a standard PCR reaction with primers F6 / B3 (see Table 2) and separated on a 4% page gel.
  • the primer pairs ISTR5 / ISTR-2 and ISTR5 / ISTR-1 were used.
  • the genomic DNAs of individual palm trees from populations of East African Tall (EAT) and Malayan Yellow Dwarf (MYD) as well as one palm tree each Rennel Island Tall (RLT) and Pemba Red Dwarf (PRD) are used as the DNAs to be examined.
  • the relevant oligodeoxynucleotides (primers) were radioactively labeled with 32 P at the end using the polynucleotide kinase in a known manner and used in a PCR reaction.
  • This is carried out in a volume of 20 ⁇ l as standard and contains 1 pmol each of the primer and 25 ng of the genomic DNA to be amplified in 1x PCR reaction buffer (eg from GIBCO / BRL), 2.5 mM MgCl 2 , 0.25 mM dNTP (deoxynucleoside Triphosphates), and 1 unit of Taq DNA polymerase.
  • 1x PCR reaction buffer eg from GIBCO / BRL
  • 2.5 mM MgCl 2 e.g., 0.25 mM dNTP (deoxynucleoside Triphosphates)
  • the mixture is first denatured for 3 minutes at 95 ° C. and then a total of 40 cycles of 95 ° C. (30 seconds, denaturation), 45 ° C. (30 seconds, addition) and 72 ° C. (2 minutes, synthesis) are carried out .
  • the reaction is ended by a synthesis step (72 ° C.
  • ISTR primers examples of oligodeoxynucleotides used for ISTR analysis
  • FIG. 6 shows a PAGE analysis of PCR products which was obtained for a total of 35 varieties or genotypes.
  • the high genetic relationship of the examined high-performance varieties can be seen from the high number of monomorphic DNA fragments. Nevertheless, a total of 44 polymorphic markers could be identified from this one analysis alone, which were mainly located in the upper region of the sequence gel. These markers were arranged in a matrix and a dendrogram was determined from them using the UPGMA method.
  • the fact that the Bonus variety (lane 33) cannot be distinguished from calcaroides-b19 (lane 32) is not surprising since this genotype is a recessive mutant generated in Bonus.
  • FIG. 7 illustrates an ISTR analysis with the primer pair ISTR5 / ISTR-2 on a cattle family (FIG. 7A) and on two sheep families with an identical father but two different mothers M1 (FIG. 7B) and M2 (FIG. 7C). From both analyzes it can be seen that 1) coconut-specific ISTR primers can also be used in the animal kingdom for fingerprint analysis, and that 2) both segregating markers (see arrows in FIG. 7C) and individual-specific markers (see stars in FIG 7) are accessible through the ISTR analysis.
  • the oligodeoxynucleotides (primers) mentioned below and listed in Table 2 were radioactively labeled at their end with 32 P using the polynucleotide kinase and used in a PCR reaction. This was carried out as standard in a volume of 20 ⁇ l and contained 1 pmol each of the primers and 25 ng of the genomic DNA to be amplified in 1x PCR reaction buffer (eg from GIBCO / BRL), 2.5 mM MgCl 2 , 0.25 mM dNTPs (deoxynucleoside Triphosphates), and 1 unit of Taq DNA polymerase. The mixture was first denatured at 95 ° C. for 3 minutes. A total of 40 cycles of 95 ° C.
  • primers F6 and B7 are contained in primers F21 and B21, respectively.
  • the results shown in FIGS. 11A and B show that overlapping primers can also be used for the ISTR analysis and that different DNA fingerprints lead to tracks 1-3).

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

Abstract

L'invention concerne l'utilisation d'amorces ou de paires d'amorces dans l'analyse d'empreintes ADN. Les amorces ou paires d'amorces permettent d'obtenir les empreintes aussi bien d'êtres humains, d'animaux, de plantes que de micro-organismes. L'invention concerne également les amorces ou paires d'amorces destinées à l'utilisation susmentionnée, et les kits contenant ces amorces ou paires d'amorces.
EP98943846A 1997-08-06 1998-08-05 Utilisation d'amorces dans l'analyse d'empreintes Withdrawn EP1002133A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP98943846A EP1002133A2 (fr) 1997-08-06 1998-08-05 Utilisation d'amorces dans l'analyse d'empreintes

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP97113601 1997-08-06
EP97113601 1997-08-06
PCT/EP1998/004877 WO1999007885A2 (fr) 1997-08-06 1998-08-05 Utilisation d'amorces dans l'analyse d'empreintes
EP98943846A EP1002133A2 (fr) 1997-08-06 1998-08-05 Utilisation d'amorces dans l'analyse d'empreintes

Publications (1)

Publication Number Publication Date
EP1002133A2 true EP1002133A2 (fr) 2000-05-24

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP98943846A Withdrawn EP1002133A2 (fr) 1997-08-06 1998-08-05 Utilisation d'amorces dans l'analyse d'empreintes

Country Status (4)

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EP (1) EP1002133A2 (fr)
AU (1) AU9159298A (fr)
CA (1) CA2300144A1 (fr)
WO (1) WO1999007885A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9002625D0 (en) * 1990-02-06 1990-04-04 Univ Singapore Human leukocyte antigen typing
CA2121696C (fr) * 1991-10-23 2003-07-08 James R. Lupski Cartographie de souches bacteriennes par amplification d'une sequence repetitive d'adn
CA2245501A1 (fr) * 1996-02-02 1997-08-07 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Utilisation d'amorces pour analyse universelle des empreintes digitales d'adn

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9907885A2 *

Also Published As

Publication number Publication date
AU9159298A (en) 1999-03-01
WO1999007885A2 (fr) 1999-02-18
WO1999007885A9 (fr) 1999-06-03
WO1999007885A3 (fr) 1999-04-29
CA2300144A1 (fr) 1999-02-18

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