EP0708782A1 - Geschlechtsspezifische dns-sonde für papageien, verfahren und geräte - Google Patents

Geschlechtsspezifische dns-sonde für papageien, verfahren und geräte

Info

Publication number
EP0708782A1
EP0708782A1 EP94922588A EP94922588A EP0708782A1 EP 0708782 A1 EP0708782 A1 EP 0708782A1 EP 94922588 A EP94922588 A EP 94922588A EP 94922588 A EP94922588 A EP 94922588A EP 0708782 A1 EP0708782 A1 EP 0708782A1
Authority
EP
European Patent Office
Prior art keywords
dna
probe
recited
seq
sex
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Withdrawn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
EP94922588A
Other languages
English (en)
French (fr)
Other versions
EP0708782A4 (de
Inventor
Siwo R. De Kloet
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.)
Florida State University
Original Assignee
Florida State University
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 Florida State University filed Critical Florida State University
Publication of EP0708782A1 publication Critical patent/EP0708782A1/de
Publication of EP0708782A4 publication Critical patent/EP0708782A4/de
Withdrawn legal-status Critical Current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
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination

Definitions

  • the present invention relates to DNA or RNA probes for binding specifically to female DNA of parrots to determine the sex of parrots within a single day and methods and kits.
  • Parrots is the collective name for approximately 350 species of birds scientifically known as the Psittacidae genus. Parrots include well-known species such as cockatoos, macaws, parakeets and a azones.
  • Parrots are among the most popular pet birds because of the ability of many species to mimic human speech and to develop strong bonds with their caretakers.
  • many species of parrots have become rare or endangered in the wild.
  • many countries have restricted or completely forbidden the exportation of parrots.
  • captive breeding has become an important procedure to prevent further depletion of wild populations and to satisfy the demand for parrots in the pet bird market.
  • the methods are not without drawbacks.
  • the external reproductive organs are physically examined to the extent
  • vent-sexing is applicable for a few bird families where the external reproductive organs are large enough that they can be observed with relative ease in the cloaca.
  • This method is, in practice, only applicable to waterfowl, such as ducks, geese, and swans, and gallinaceous birds, such as chickens, where the method can be applied at an early age.
  • waterfowl such as ducks, geese, and swans
  • gallinaceous birds such as chickens
  • a second method which is an invasive method, involves the examination of internal reproductive organs.
  • a bird must be placed under general anaesthesia and an incision made so that the internal reproductive organs can be observed with an endoscope.
  • an endoscope Although this is the most often used procedure, it causes great trauma to the bird and often results in infection.
  • the method cannot be used with very young birds, because of their sensitivity to surgery, and the insufficient development of the internal reproductive organs in such young birds.
  • a third method involves biochemical determinations of the concentrations of the steroid sex hormones, estradiol and testosterone, from blood and/or faecal matter.
  • This procedure is based on the observation that male animals are characterized by a high concentration of testosterone in their blood, while females have high levels of estradiol.
  • a disadvantage of this procedure is that it is technica ⁇ -ly very complicated and requires experienced personnel for its operation.
  • Another problem is that the procedure has no diagnostic value when used on young animals having low steroid hormone concentrations.
  • the method can be applied to faecal matter rather than blood samples, it is often difficult to assign faecal samples to individuals birds when more than a few individual birds are under observation.
  • the fourth method which is also an invasive method, is based on the occurrence of a specific chromosome in female birds.
  • Male birds have two Z sex chromosomes, "ZZ,” whereas female birds have one W sex chromosome and one Z sex chromosome ("Z ”) .
  • the chromosome has some unique staining characteristics and can, therefore, be observed and distinguished by microscopic examination of bird chromosome preparations.
  • a disadvantage is that an expensive laboratory equipment is needed and highly qualified personnel are required for the performance of this procedure.
  • an unambiguous identification of the sex chromosome is not possible.
  • the fifth method available today is a fingerprint based DNA analysis which involves the identification of sex specific DNA fragments by DNA fingerprinting.
  • the DNA of individual birds is degraded with specific enzymes, resulting in the generation of specific fragments, which upon fractionation by electrophoresis forms a pattern specific for each individual bird, including its sex.
  • the disadvantage of a fingerprint based DNA analysis is that it is technically very complicated and takes approximately one week to perform. Moreover, it requires isolation of very pure DNA, enzymatic digestion of the isolated DNA, fractionation by electrophoresis of the DNA fragments in the digest, and transfer of these fragments from the gel to a nylon membrane, followed by probing with a specific probe. Moreover, radioactive labelling of the probe is essential to the procedure, thus requiring the use of highly sophisticated equipment and facilities.
  • the present invention overcomes certain of the above-mentioned problems and shortcomings of the present state of the art through the discovery of novel nucleotide sequences derived from the*-W chromosome of a female parrot, the African grey parrot, Psittacus erithacus.
  • novel nucleotide sequences of the present invention are highly female specific for this species of parrot. Moreover, they are believed to be highly female specific for almost all other species of parrot.
  • the novel, universal nucleotide sequences are used as DNA or RNA probes in a quick and reliable procedure for sex determination of parrots.
  • the sex-typing procedures of the present invention can be completed within 24 hours.
  • the procedures of the present invention are reliable because virtually all species of parrots contain a sex specific component or components on the sex chromosome which is closely similar to the sex specific component, i.e., the nucleotide sequences, isolated from the African grey parrot in accordance with the present invention.
  • the present invention requires only a few microliters of blood, which can be collected easily without anaesthesia, from a wing vein or a toe of the parrot.
  • the blood sample may be acquired from a blood feather such as a developing secondary or primary flight or tail fea * feher.
  • a blood sample is first obtained from a parrot of choice, and DNA is then obtained from this blood sample (10 - 60 min) .
  • the DNA is denatured, bound to a nylon membrane and prehybridized (1-2 hrs) .
  • the DNA bound to the nylon membrane is than hybridized with a radioactive or nonradioactive DNA probe solution (12 hrs) of the present invention, and washed to remove the non-specifically bound DNA probe (1 hr) .
  • the specifically bound DNA probe is then visualized with the appropriate procedure (2 hrs) to determine the sex of the parrot.
  • the DNA or RNA probes used in the present invention are sex specific but not parrot-species specific. Radioactively or nonradioactively (biotin) tagged DNA or RNA probes of the present invention are successful in determining the sex of many parrot species in avian collections.
  • the advantages of using the DNA or RNA probes, methods and kits of the present invention for the determination of the sex of parrots include, for example, 1) rapid determination (24 hrs) ; 2) major surgical procedures are not required; 3) the use of blood as a readily available source of DNA; 4) the use of a safe, stable, highly sensitive and highly sex specific but not species-specific DNA probe; 5) the use of simple procedures utilizing standard clinical and research laboratory equipment which require minimal technical expertise for their operation; and 6) technical simplicity as compared to currently available procedures.
  • the procedures of the present invention can be easily practiced by veterinarians and breeders in their offices and by other qualified personnel.
  • the present invention is believed to provide a solution to the sex-typing parrot art that has long sought rapid and reliable methods for determining the sex of different species of parrots.
  • This is accomplished by the present invention, as indicated above, through the identification of novel, universal nucleotide sequences which are useful as DNA or RNA probes that are complementary to DNA segments on the chromosome which are characteristic for female parrots of the Psittacidae genus.
  • Applications of the DNA or RNA probes contemplated by the present invention therefore include, for example, determination of the sex of parrots in captive breeding programs.
  • the applications of the probes include the determination of the sex of parrots in wild populations as part of, for example, research and ecological studies.
  • FIGS. in which are shown characteristics corresponding to the novel nucleotide sequences of the present invention from which certain of their features and advantages will be apparent:
  • FIG. 1 describes the nucleotide sequences of the two strands of the 461 bp sex specific DNA repeat of the African grey parrot.
  • a randomly chosen second clone of the repeat shows 17 nts or 4% difference with the first clone.
  • FIG. 2 describes the structure of one of the strands of the 461 nt sex specific repeat of the
  • T oligo dT fragments of 2-6 thymine
  • novel nucleotide sequences of this invention preferably used as DNA or RNA probes, have been derived from the DNA of the female African grey parrot Psittacus erithacus. More particularly, they have been cloned as a fragment of 461 base pairs, obtained with the restriction nuclease Mspl, in the vector pGEM3Z+ and the host Eschericia coli 31/17.
  • the 461 base pair repeat is derived and isolated from
  • the DNA component is tandemly repeated with a copy number of approximately 12,000 copies per female African grey parrot genome and forms a substantial part of the W chromosomal DNA of this species.
  • the nucleotide sequence of the component is conserved among the DNA of females of many other species of parrot. While the 461 base pair fragment has been sequenced as reported in FIG. 1, the remaining base pairs in the 570 base pair fragment from which the 461 base pair fragment has been derived and isolated have not been sequenced.
  • the novel procedure to demonstrate the presence of female W chromosome specific components in parrot DNA utilizes a probe comprising a trace amount of radio labelled female parrot DNA and a large excess (4000 fold) of unlabelled male DNA in an analysis of enzymatically digested and electrophoretically fractionated female parrot DNA.
  • the excess unlabelled male DNA acts to dilute the radio-label in the components which are common to male and female DNA, so that common sequences do not produce any signal in the analysis, and the only signal observed is that. * - produced by female specific components.
  • Mspl Utilizing this procedure with the restriction enzyme Mspl, in a digest of the female African grey parrot, Psittacus erithacus. identification of a component of approximately 450 base pairs, which does not occur in the male DNA, is accomplished.
  • This fragment is isolated from the electrophoretically fractionated Mspl digest and cloned in plasmid vector .
  • pGEMZ+ in the bacterium Eschericia coli 81/17. This cloned component has been used for the determination of the nucleotide sequence of the female specific component, its copy number and genomic organization, and the conservation among other species of parrots.
  • FIG. 1 shows that the sequence of the component has a molecular length of 461 base pairs.
  • FIG. 2 shows that the fragment has an internal repeat structure in which groups of 2 - 6 thymine residues are repeated an average of 10.5 nucleotides. This sequence characteristic is typical for a rather unusual so called "curved DNA element”.
  • a curved DNA is a double stranded (native) DNA which most often has short runs of 2-6 adenine or thymine residues at an average distance of 10.5 nts, which is just a complete winding of the DNA double helix.
  • the curved nature of such DNA has been demonstrated by electronmicroscopy and circularization experiments. Curved DNA exhibits an anomalous, slow electrophoretic velocity in polyacrylamide gels. This latter characteristic is usually taken as diagnostic evidence for the curved structure.
  • the curved structure is an inherent property of such DNA and should be distinguished from so called bent DNA. The latter owes its curvature to interaction with certain proteins.
  • DNA components can occur either as unique or as repeated sequence elements.
  • a quantitative analysis shows that the sex specific component amounts to approximately 0.3% of the genome of the female African grey parrot, whereas it amounts to at most 0.005% of the male genome. See Table I.
  • the female African grey parrot genome contains about 12,000 copies of the sex specific DNA component of a length of 463 base pairs. These copies are tandemly organized in the genome.
  • In situ hybridization to chromosomes of the African grey parrot shows strong hybridization of the DNA or RNA probes of the present invention to the W chromosome in the female whereas such hybridization is not observed in the male.
  • the DNA probes of the present invention can be produced by chemical synthesis, recombinant or cloning technology or any other methods available in the art so long as the methodology selected does not interfere with their utilities stated herein. Moreover,- the DNA probes of the present invention may be modified by adding, deleting and/or substituting nucleotides to form DNA probes of varying lengths which are functionally equivalent to the nucleotide sequences set forth in FIG. 1. In addition, RNA probes with nucleotide sequences contemplated by the present invention may be substituted for the DNA probes. Therefore, it is to be understood by those versed in this art that any DNA or RNA nucleotide sequence, including equivalents and active segments of the nucleotide sequences depicted in FIG.
  • active nucleotide fragments in accordance with the present invention are those fragments derived by, for example, digesting the 461 base pair fragment with the enzyme SAU3A1 at a GATC site using digestion techniques known to those skilled in the art.
  • SAU3A1 the 461 base pair fragment
  • - fragment is cleaved at the GATC sites beginning with the nucleotide designated as 245 in the 5'-3' sequence and with the nucleotide designated as 213 in the 3'-5' sequence in FIG. 1 to generate four active fragments having, for example, bases corresponding to bases 1-248 and 249-461 in the 5'-3' sequence and corresponding to bases 1-216 and 217-461 in the 3'-5' sequence as designated in FIG. 1.
  • the DNA or RNA probes of the present invention may be formed into kits which can be easily used by, for instance, veterinarians, breeders, as well as other qualified personnel interested in sex-typing parrots of the Psittacidae genus.
  • a typical kit in accordance with this invention includes blood sample stabilizing Solution A; proteinase-K and sodium sarcosylate Solutions, or a commercially available fast DNA isolation kit; DNA denaturing and neutralizing Solutions; nylon membranes; biotin-labelled sex specific DNA or RNA probe; hybridization Solution B; wash Solutions C and D; and a detection system specific for the biotin tag so that all that is required to carry out the sex-typing methods of the present invention is standard laboratory equipment.
  • blood is used as a source of DNA because avian erythrocytes are nucleated and contain DNA.
  • Avian blood contains on average approximately 5 to 10 mgs of DNA per ml.
  • blood samples approximately 25 to 100 ⁇ l, are obtained by brachial (wing) vein puncture or by clipping of a toe nail or by removing a blood feather such as a secondary, or primary flight feather or a tail feather. Clotting of the blood is prevented by immediate dilution, after collection, with an equal volume of a solution containing about 0.15M NaCl and about 0.05 M sodium EDTA, pH 7.5 (Solution A). In this solution, blood samples can be stored for at least a week in a refrigerator at 4*c.
  • the major contaminant of the DNA in avian blood is protein, which can be removed from the sample by protease digestion, e.g., phenol, or chloroform extraction in the presence of (cationic or anionic) detergents.
  • protease digestion e.g., phenol
  • chloroform extraction the chloroform should be removed by for example ⁇ l of the 1:1 average 50 mgs of DNA
  • solution A is diluted with 90 ⁇ l of Solution A and incubated for one hour at about 65*C with proteinase K (2 ⁇ gs per 100 ⁇ l) and sodium lauryl sarcosylate (0.2%).
  • a commercially available DNA isolation system e.g. from Invitrogen, 3985 Sorrento Valley Blvd, San Diego, CA 92121 or Washington Biotechnology Inc., 6917 Arlington Rd. , Bethesda, MD 20814) can be used for this purpose.
  • DNA samples obtained are then denatured with NaOH (final concentration about 0.5 M) and neutralized with about 1 M NaH 2 P0 .
  • Samples containing approximately 1 mg of denatured parrot DNA are then loaded and immobilized onto nylon filters (0.2 ⁇ M pore size) with the aid of a slot or dot blot apparatus.
  • the filters are then dried in vacuo at about 80°C for about two hrs to bind the DNA irreversibly to the filters.
  • filters containing the DNA samples Prior to hybridization with the probe, filters containing the DNA samples are prehybridized for about one hr at about 65 ⁇ C in a solution containing about 0.9 M NaCl, 0.1 M Tris-HCl buffer pH 7.8, 0.05 M Na 2 EDTA, 0.2% sodium lauryl sulphate and 500 ⁇ g per ml heparin as a blocking agent (Solution B).
  • the sex specific DNA fragment obtained by cloning from the genome of the female African grey parrot as described above and recited in FIG. l, is tagged by enzymatic or nonenzymatic procedures with radioactive P 32 or nonradioactive biotin or fluorescent groups like fluoresceine or rhodamine using technologies available to these in the labeling art.
  • the DNA probe may also be tagged with fluorescent dyes, such as fluoreseine or rhodamine using techniques known to those versed in this art.
  • the DNA probe is tagged by, e.g., labelling with biodUTP using nick translation or random primer extension.
  • an RNA probe it is labelled with, e.g.
  • bioUTP using TT- RNA polymerase (pGEM3Z+ has a T7 RNA polymerase promoter site) .
  • streptavidine and avidine are proteins which have a ver yhigh specific affinity for biotein; phosphatase as an enzyme which hydrolyes phosphate esters.) and the subsequent hydrolysis of a so-called chromogenic substrate which consists of an uncoloured phosphate ester which becomes strongly coloured after removal of the phosphate by the phosphatase.
  • esters are bromo-indoxyl phosphate which is uncoloured, but which generates dark blue indigo after removal of the phosphate.
  • Chemiluminescent detection systems make use of chemiluminescent substrates in a similar phosphatase catalyzed reaction, followed by photographic detection of the emitted light.
  • a southern blot of the gel is made and probed with a genomic DNA probe made from 25 ng radioactively (P 32 ) labelled female DNA [labelled by random primer extension, Feinberg, A.P. et al.: Anal. Biochem.. 132:6-13 (1983), preannealed to 100 ⁇ g reiterated, denatured male African grey parrot Cot2 DNA (Cot indicates a degree of repetitivity of the DNA -sample, see: De Kloet pH and de Kloet SR (1992) Molecular determination of the sex of parrots (FOCUS (BRL) 14:106-108 (1992)).
  • a preparative (larger) gel is run and the gel slice containing the 461 bp sex specific fragment cut out.
  • the sex specific fragment is isolated from the gel by the powdered glass procedure, Vogelstein, B. et al.: Proc. Natl. Acad. Sci. USA. 76:615-619 (1979).
  • the fragment is ligated into the powdered glass procedure. See Vogelstein B. et al.: Proc. Natl. Aca. Sci. USA. 76:615-619 (1979).
  • the fragment is ligated into the Accl site of the plasmid pGEM3Z+ and the ligation product is used to transform E.
  • the white colonies (containing plasmids with inserts) are selected and transferred to fresh FL plates covered w-ith a Nylon filter. Control plates without a filter are also inocculated with the colonies in the same pattern. After growth until the colonies are detectable, the nylon filter is removed from the plate and dried in a vacuum oven at 80°C. Colonies with a sex specific insert are identified by colony hybridization (Maniatis, T. et al. (1982) "Molecular cloning; a laboratory manual.” Cold Spring Harbour Laboratory.
  • 100 ⁇ l (corresponding to 5 ⁇ l of blood) is taken and diluted with 200 ⁇ l of a solution containing 8% sodium dodecyl trimethylammonium bromide, 1.5 M NaCl, 100 mM Tris-HCl buffer pH 8.6, 50 mM EDTA. After heating for two minutes at 68°C, vortexing for two minutes with 300 ⁇ l of chloroform and centrifugation for two minutes in an Eppendorf centrifuge, the aqueous phase (top layer) (250 ⁇ l) is collected.
  • this aqueous phase contains approximately 25 (5 x 5) ⁇ g of DNA per 250 ⁇ l, or 1 ⁇ g of DNA per 10 ⁇ l. Fifty ⁇ l of the aqueous phase is then taken (containing approximately 5 ⁇ g of DNA) , diluted with 50 ⁇ l to TE (0.01 M Tris-HCl buffer pH 8.0, 0.001 M Na 2 EDTA) , and the DNA is denatured (make single stranded) by the addition of 100 ⁇ l of 1 M NaOH.
  • the denatured DNA is neutralized with 200 ⁇ l 1 M NaH 2 P0 4 and 100 ⁇ l of 1.5 M NaCl is added to a final volume of 500 ⁇ l.
  • 100 ⁇ l of this solution (containing approximately 1 ⁇ g of DNA) is applied to a Nylon filter with the aid of a slot blot or dot blot apparatus. The filter is dried and baked in an oven at 80°C.
  • the filter with the bound denatured DNA is then preincuba ed for thirty minutes at 60"C with 0.2 ml per cm 2 of a solution containing 0.9 M NaCl, 0.002 M Na EDTA, 0.02 M Tris-HCL pH 8.0, 0.1% sodium dodecyl sulphate and 500 ⁇ g per ml of heparin (as the blocking agent to prevent direct binding of the probe to the filter without binding to the DNA) and 1000 ⁇ g per ml of sodium pyrrophosphate.
  • Heat denatured radioactively or nonradioactively (biotin) labelled parrot sex specific probe (40 - 100 ng per 10 ml buffer) is then added and the incubation continued overnight.
  • the filters are washed with 1 x SSC to remove the unhybridized probe, and the bound radioactive label visualized by autoradiography or a betascope.
  • the biotin labelled probe is visualized with any of the appropriate techniques like streptavidine - alkaline phosphatase etc.
  • the procedure is as follows. A growing primary or secondary feather is collected and the featherpulp is squeezed out of the feather into 1 or 2 ml (depending on the size of the feather) of buffer A (see above) or any other stabilizing solution (Dulbecco s modified Eagles etc.). To this suspension is added 75-150 ⁇ l of a solution of 10 ⁇ g per ml collagenase (Closteridium haemolyticum) in water and the mixture incubated for two hours at 37°C. After gentle homogenization DNA is prepared from the ⁇ resulting cells as described above for blood DNA. After measuring the amount of prepared DNA with a spectrophotometer, the DNA is again alkali denatured, applied to a nylon filter and the filter prehybridized and hybridized with a probe as described above.
  • buffer A see above
  • any other stabilizing solution Dulbecco s modified Eagles etc.
  • FIG. 3 shows that in a blotting experiment, DNA of the African grey parrot as well as of other species of parrots, belonging to the different major subfamilies of the Psittacidae. namely, the conure, the cockatoo, the lory, the amazone and the macaw also produce a sex specific signal with the radioactively labelled female specific DNA component of the African grey parrot, showing that the sex specific characteristics of the component is conserved among the parrot family.
  • different amounts of DNA of males and females of the species indicated are immobilized on a nylon filter and hydribized with the p 32 -labelled African grey parrot sex specific DNA repeat element as the probe.
  • the sex specific hybridization of this probe with DNA of all six species shows that the sex specific DNA component is structurally conserved between the different species and can be used for the determination of all species.
  • MOLECULE TYPE DNA (genomic)
  • TCTAACCCAT TTTTTCAACA GTTCTAGCTC GGTTTAAGTA GTTTTTGCTT TTTTCTAACC
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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)
EP94922588A 1993-07-15 1994-07-15 Geschlechtsspezifische dns-sonde für papageien, verfahren und geräte Withdrawn EP0708782A4 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US93198 1987-09-04
US9319893A 1993-07-15 1993-07-15
PCT/US1994/008023 WO1995002605A1 (en) 1993-07-15 1994-07-15 Sex-specific dna probe for parrots, methods and kits

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EP0708782A1 true EP0708782A1 (de) 1996-05-01
EP0708782A4 EP0708782A4 (de) 1997-01-02

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EP (1) EP0708782A4 (de)
AU (1) AU7364594A (de)
CA (1) CA2167295A1 (de)
WO (1) WO1995002605A1 (de)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPN283195A0 (en) * 1995-05-05 1995-06-01 Australian Water Technologies Pty Ltd Method for the detection of viable cryptosporidium parvum cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NUCLEIC ACIDS RESEARCH, vol. 20, no. 19, 11 October 1992, OXFORD GB, pages 5235-5236, XP002017078 C.Y.MIYAKI ET AL.: "Sex Typing of Aratinga Parrots Using the Human Minisatellite Probe 33.15." *
See also references of WO9502605A1 *

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CA2167295A1 (en) 1995-01-26
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WO1995002605A1 (en) 1995-01-26

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