CA2167295A1 - Sex-specific dna probe for parrots, methods and kits - Google Patents
Sex-specific dna probe for parrots, methods and kitsInfo
- Publication number
- CA2167295A1 CA2167295A1 CA002167295A CA2167295A CA2167295A1 CA 2167295 A1 CA2167295 A1 CA 2167295A1 CA 002167295 A CA002167295 A CA 002167295A CA 2167295 A CA2167295 A CA 2167295A CA 2167295 A1 CA2167295 A1 CA 2167295A1
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- CA
- Canada
- Prior art keywords
- dna
- probe
- parrot
- recited
- oligonucleotide probe
- 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.)
- Abandoned
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Classifications
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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
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
Abstract
Novel DNA probes for determining the sex of parrots in captive breeding programs and in wild populations are disclosed. The unique DNA probes of the present invention are sex specific, but not parrot-species specific. In addition, the DNA probes of the present invention are reliable, yield results within approximately 24 hours, easy to use, and are not harmful to the parrots being tested.
Description
V095/02605 2 1 ~ 7 2 9 5 PCT~S94/080~
SEX~ lC DNA PROBE FOR PARROTS, METHODS AND KITS
Field of the Invention 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.
Bac~ r ~
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 amazones.
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. However, as a result of habitat loss and the pet bird trade, many species of parrots have become rare or endangered in the wild.
WO 95/02605 PCT/US94/08r G;~ , 6 '`Z ~ 9 In order to protect populations of wild parrots, many countries have restricted or completely forbidden the exportation of parrots. Moreover, 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.
For such breeding projects, male and female birds are needed. Therefore, the ability to determine the sex of individual birds is of major importance. Unfortunately, most species of parrots are monomorphic, meaning that there are no recognizable visible or audible differences between male and female birds. More specifically, virtually all juveniles and about 60% of the adults of all avian species are monomorphic.
Presently, there are basically five methods relied upon today to determine the sex of birds.
Unfortunately, the methods are not without drawbacks. In one method, the external reproductive organs are physically examined to the extent -possible. This method, called "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 -~095l0~05 ~1 6 7 2 9 5 PCT~S94/08023 applied at an early age. However, the difficulty in handling, as well as the difficulties in observing the external reproductive organs, make '~vent-sexing~
unsuitable for use with parrots, as well as other s birds in which the external reproductive organs cannot be observed.
A second method, which is an invasive method, involves the examination of internal reproductive organs. According to this method, a bird must be placed under general anaesthesia and an incision made so that the internal reproductive organs can be observed with an endoscope. Although this is the most often used procedure, it causes great trauma to the bird and often results in infection. Moreover, 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 WO95l02605 PCT~S94/080 ~,~G~I ~9~
personnel for its operation. Another problem is that the procedure has no diagnostic value when used on young animals having low steroid hormone concentrations. Although 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 W 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 ("ZW"). In many, but not all bird species, the W 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. As a further drawback associated with this method, in a number of bird species, including some parrots, an unambiguous identification of the W sex chromosome is not possible.
The fifth method available today, is a fingerprint based DNA analysis which involves the identifieation of sex specific DNA fragments by DNA
`~095/02605 ~1 ~ 7~9 ~ PCT~S94/08023 fingerprinting. According to this method, 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.
In view of the foregoing, it is evident that there is a demand for a simple, quick, and accurate method to determine the sex of birds, such as parrots.
SummarY of the Invention In brief, 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 WO95/0~05 ~t6~ 2 PCT~S94/080 grey parrot, Psittacus erithacus. Uniquely, the 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.
According to the present invention, the novel, universal nucleotide sequences are used as DNA
or RNA probes in a quick and reliable procedure for sex determination of parrots. Unique to this procedure and as an advantage over prior invasive procedures utilized heretofore, the sex-typing procedures of the present invention can be completed within 24 hours. Moreover, the procedures of the present invention are reliable because virtually all species of parrots contain a sex specific component or components on the W 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.
In carrying out the present invention, it requires only a few microliters of blood, which can be collected easily without anaesthesia, from a wing vein or a toe of the parrot. As an alternative, the blood sample may be acquired from a blood feather such as a developing secondary or primary flight or tail fea~her.
vos5lo~os ~ PCT~S94/080~
21 ~ 7 To summarize a procedure of the present invention, 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 ~1~729~ !S '' AUG 1996 laboratory equipment which require minimal technical ex^pertise for their operation; and 6) technical simplicity as compared to currently available procedures. Thus, the ~,oce~res of the present invention can be easily practiced by veterinarianS
and breeders in their offices and by other qualified personnel.
Accordingly, it should now be appreciated that the present invention is believed to provide a solution to the s-x-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 seguences which are useful as DNA or RNA
probes that are complementary to DNA segments on the w chromosome ~h~ re characteristic for female parrots o~ P~ us genus. Applications of the DNA or RNA p.-~obes contemplated by the present invention therefore include, for example, determination of the sex of parrots in captive breeding programs. Moreover, 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.
The above features and advantages will be better understood with reference to the FIGS., A~dElYDE~
~5 ~1 ~ 7295 PCT~594/o8 Detailed Description and Examples set out hereinbelow. It will also be understood that the biological materials, methods and kits of this invention are exemplary only and are not to be regarded as limitations of this invention.
Brief DescriPtion of the FIGS.
Reference is now made to the accompanying 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 African grey parrot in order to illustrate that it is made of a linear array of subfragments in which oligo dT fragments of 2-6 thymine (=T) residues are repeated at an average distance of 10.5 nts, which represents one complete turn of the DNA double helix.
FIG. 3 describes the signals obtained by hybridizing the cloned p32-labelled African grey parrot sex specific DNA repeat element with three differen~ amounts of nylon immobilized male and W095/02605 PCT~S94/08C
~6~ ~9~
female species: 1 = chattering lory (Lorius garulus);
SEX~ lC DNA PROBE FOR PARROTS, METHODS AND KITS
Field of the Invention 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.
Bac~ r ~
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 amazones.
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. However, as a result of habitat loss and the pet bird trade, many species of parrots have become rare or endangered in the wild.
WO 95/02605 PCT/US94/08r G;~ , 6 '`Z ~ 9 In order to protect populations of wild parrots, many countries have restricted or completely forbidden the exportation of parrots. Moreover, 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.
For such breeding projects, male and female birds are needed. Therefore, the ability to determine the sex of individual birds is of major importance. Unfortunately, most species of parrots are monomorphic, meaning that there are no recognizable visible or audible differences between male and female birds. More specifically, virtually all juveniles and about 60% of the adults of all avian species are monomorphic.
Presently, there are basically five methods relied upon today to determine the sex of birds.
Unfortunately, the methods are not without drawbacks. In one method, the external reproductive organs are physically examined to the extent -possible. This method, called "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 -~095l0~05 ~1 6 7 2 9 5 PCT~S94/08023 applied at an early age. However, the difficulty in handling, as well as the difficulties in observing the external reproductive organs, make '~vent-sexing~
unsuitable for use with parrots, as well as other s birds in which the external reproductive organs cannot be observed.
A second method, which is an invasive method, involves the examination of internal reproductive organs. According to this method, a bird must be placed under general anaesthesia and an incision made so that the internal reproductive organs can be observed with an endoscope. Although this is the most often used procedure, it causes great trauma to the bird and often results in infection. Moreover, 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 WO95l02605 PCT~S94/080 ~,~G~I ~9~
personnel for its operation. Another problem is that the procedure has no diagnostic value when used on young animals having low steroid hormone concentrations. Although 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 W 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 ("ZW"). In many, but not all bird species, the W 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. As a further drawback associated with this method, in a number of bird species, including some parrots, an unambiguous identification of the W sex chromosome is not possible.
The fifth method available today, is a fingerprint based DNA analysis which involves the identifieation of sex specific DNA fragments by DNA
`~095/02605 ~1 ~ 7~9 ~ PCT~S94/08023 fingerprinting. According to this method, 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.
In view of the foregoing, it is evident that there is a demand for a simple, quick, and accurate method to determine the sex of birds, such as parrots.
SummarY of the Invention In brief, 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 WO95/0~05 ~t6~ 2 PCT~S94/080 grey parrot, Psittacus erithacus. Uniquely, the 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.
According to the present invention, the novel, universal nucleotide sequences are used as DNA
or RNA probes in a quick and reliable procedure for sex determination of parrots. Unique to this procedure and as an advantage over prior invasive procedures utilized heretofore, the sex-typing procedures of the present invention can be completed within 24 hours. Moreover, the procedures of the present invention are reliable because virtually all species of parrots contain a sex specific component or components on the W 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.
In carrying out the present invention, it requires only a few microliters of blood, which can be collected easily without anaesthesia, from a wing vein or a toe of the parrot. As an alternative, the blood sample may be acquired from a blood feather such as a developing secondary or primary flight or tail fea~her.
vos5lo~os ~ PCT~S94/080~
21 ~ 7 To summarize a procedure of the present invention, 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 ~1~729~ !S '' AUG 1996 laboratory equipment which require minimal technical ex^pertise for their operation; and 6) technical simplicity as compared to currently available procedures. Thus, the ~,oce~res of the present invention can be easily practiced by veterinarianS
and breeders in their offices and by other qualified personnel.
Accordingly, it should now be appreciated that the present invention is believed to provide a solution to the s-x-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 seguences which are useful as DNA or RNA
probes that are complementary to DNA segments on the w chromosome ~h~ re characteristic for female parrots o~ P~ us genus. Applications of the DNA or RNA p.-~obes contemplated by the present invention therefore include, for example, determination of the sex of parrots in captive breeding programs. Moreover, 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.
The above features and advantages will be better understood with reference to the FIGS., A~dElYDE~
~5 ~1 ~ 7295 PCT~594/o8 Detailed Description and Examples set out hereinbelow. It will also be understood that the biological materials, methods and kits of this invention are exemplary only and are not to be regarded as limitations of this invention.
Brief DescriPtion of the FIGS.
Reference is now made to the accompanying 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 African grey parrot in order to illustrate that it is made of a linear array of subfragments in which oligo dT fragments of 2-6 thymine (=T) residues are repeated at an average distance of 10.5 nts, which represents one complete turn of the DNA double helix.
FIG. 3 describes the signals obtained by hybridizing the cloned p32-labelled African grey parrot sex specific DNA repeat element with three differen~ amounts of nylon immobilized male and W095/02605 PCT~S94/08C
~6~ ~9~
female species: 1 = chattering lory (Lorius garulus);
2 = cherry-headed conure (Aratinga erythrogenys); 3 =
sulphur-crested cockatoo (Cacatua galerita); 4 blue-fronted amazon (Amazona aestiva); 5 = blue and gold macaw (Ara ararauna); 6 = African grey parrot (Pisttacus erithacus).
Detailed Descri~tion By way of illustrating and providing a more complete appreciation of the present invention and many of the att~n~t advantages thereof, the following detailed description is provided concerning the novel nucleotide sequences, methods and kits.
The 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 MspI, in the vector pGEM3Z+ and the host Eschericia coli 31/17.
The 461 base pair repeat is derived and isolated from -a base pair repeat of about 570 base pairs and is made up of 43 subfragments in which oligo dT
fragments of 2-6 thymine residues are repeated at an average distance of lO.Sbp. More specifically, the 2-6 long oligo T fragments are part of 43 subfragments of a minimum length of about 4 nts and a maximum ~ength of about 20 nts, and an average length ~095/0~05 PCT~S94/080~
216729~
of about 10.5 nts which together form the 461 nt long fragment which is cloned. This latter fragment is again tandemly repeated 12,000 times. 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.
FOCUS (BRL) 14: 106-108 (1992). 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.
W095/0~05 ~6~ 295 PCT~S94/08 Utilizing this procedure with the restriction enzyme MspI, 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 MspI 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 circular~zation experiments. Curved DNA exhibits an vo 95~0260s 21 6 7295 PCT~S94/080~
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. Hence, 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 F the DNA probes of the present invention may ~167~?9S- PCT~JS 94/0~423 IP~ JS 3 :AUG 1995 be modified by adding, deleting and/or substitutingnucleotides 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. 1, which is complimentary to a DNA~segment on a W chromosome which is characteristic for a female parrot of the Psittacus genus is contemplated by the present invention. Examples of active nucleotide fragments in accordance with the present invention are those fragments derived by, for example, digesting the 461 base pair fragment with t.~ el,zyme SAU3Al at a GATC
site using diges~ion t.~hn.i ~ known to those skilled in the art. When` ~h~ 461 base pair fragment is digested with the enzyme SAU3Al, the 461 base pair 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 co~e~onding to bases 1-248 and 249-461 in the 5'-3' sequence and _~9~J
2167,~95 - PC~IS 94/08 023 IPEAJUS ~}~AUG ~
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 gualified personnel interested in sex-typing parrots~ of the Psittacus genus. A
typical kit in accordance with this invention includes blood sample stabilizing Solution A;
lo proteinase-K and sodium sarcosylate Solutions, or a commercially available fast DNA isolation kit; D~A
denaturing and neutralizing Solutions; nylon membranes; biotin-labelled sex specific DNA or RNA
probe; hybridization Solution ~: 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 pres~nt invention is standard laboratory e~uipm~t.
According to the prese~lt invention, 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.
In carrying out the sex typing of parrots in accordance with the present invention, blood samples, approximately 25 to 100 ~1, 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 ,~D ~r Yo 95,~.605 ~ 1 6 7 2 9S PCT~S94/08023 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. ~When chloroform extraction is used, the chloroform should be removed by for example centrifugation.~ Accord~ 1 of the 1:1 diluted blood sample ~(containing on average 50 mgs of DNA) is diluted with 90 ~1 of Solution A and incubated for one hour at about 65-C with proteinase K (2 ~gs per 100 ~1) 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 NaH2PO4. Samples containing approximately 1 mg of denatured parrot DNA
VOg5/02605 PCT~S94/080~
~` 2~1 6729~
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.
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, O.l M Tris-HCl buffer pH
7.8, 0.05 M Na2EDTA, 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. 1, is tagged by enzymatic or nonenzymatic procedures with radioactive p32 or nonradioactive biotin or fluorescent groups like fluoresceine or rho~mine using technologies available to these in the labeling art. It should be understood that the DNA probe may also be tagged with fluorescent dyes, such as fluoreseine or rhodamine using techni~ues known to those versed in this art. In the present example the DNA probe is tagged by, e.g., labelling with biodUTP
using nick translation or random primer extension.
As to an RNA probe, it is labelled with, e.g., bioUTP
using T~ RNA polymerase (pGEM3Z+ has a T7 RNA
WO 9S/02C05 ; Z l 6 ~ PCT~S94/080 polymerase promoter site). The tagged, denatured DNA
probe is then incubated for approximately 60 - 65-C
and at a concentration of about 25 ng per ml of Solution B with the nylon filters containing the parrot DNA. Subsequent to incubation, the filters are washed first at room temperature with 1 x SSC (=
0.15 M NaCl, 0.015 M sodium citrate pH. 7.0, Solution C) (five times for about five minutes with about lO0 ml) and subse~uently one time for about two minutes at about 55-C with approximately 100 ml 1 x SSC
containing about 0.1% sodium lauryl sulphate (Solution D). Binding of radioactively labelled probe is visualized and quantified by autoradiography and/or a betascope. In this Example, bound probes labelled nonradioactively with biotin are visualized through a commercially available detection process which involves a specific color reaction or chemiluminescence. The specific colour reaction involves, e.g., the binding of a streptavidin or avidin phosphatase conjugate to the biotin residue -(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 ~hosphate by the phosphatase. Examples of VOg5/0~05 t 6 7~9 ~ PCT~Sg4tO8023 such 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.
The present invention will now be further illustrated with reference to the following Examples.
ExamPle I
SEX ~r.~ lC DNA F'177'r~F~IT
Female african grey parrot DNA (10 ug) is digested in 100 ~1 of a buffer (containing 6 mM
MgC12, 6 mM Tris-HCl buffer pH 7.5, 50 mM NaCl) with 20 units of the restriction endonuclease Mspl for 3 hrs at 37- C. For identification purposes of the sex specific DNA fragment, 10 ~1 of the digest is fractionnated by electrophoresis through a 1% agarose gel in TPE buffer (TPE = 0.08 M Tris-phosphate pH
7.5, 0.008 M EDTA). A southern blot of the gel is made and probed with a genomic DNA probe made from 25 ng radioactively (p32) labelled female DNA [labelled by random primer extension, Feinberg, A.P. et al.:
Anal. Biochem., 132:6-13 (1983), preannealed to 100 2 5 ~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
W095l02605 PCT~S94/080 2 g ~
(1992) Molecular determination of the sex of parrots (FOCUS (BRL) 14:106-108 (1992)).
After identification of the sex specific DNA fragment by autoradiography or with a betascope, 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 ACC1 site of the plasmid pGEM3Z+ and the ligation product is used to transform E. coli 71/18 using the CaC12 procedure (Maniatis, T. et al.
(1982) "Molecular cloning; a laboratory manual."
Cold Spring Harbour Laboratory. Cold Spring Harbour, NY, 2) probe labelling with biotin:). The transformed bacteria are spread on 20 ml agar plates containing Luria bertani (L8) medium (LB = 1%
bacto=tryptone, 1% NaCl, 0.5% bacto yeast extract pH
7.4) containing per ml 50 ~gs of penicillin, 150 ~gs bromo-idnolyl-beta-galactoside and 75 ~g isopropyl beta thiogalactoside. After growth vernight at 37-C, the white colonies (containing plasmids with inserts) are selected and transferred to fresh FL plates covered ~ith a Nylon filter. Control plates without ~095t02605 1 6 729 ~ PCT~S94/08023 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. Cold Spring Harbour, NY, 2) probe labelling with biotin:) using the same genomic sex lo specific DNA probe as is used initially for determination of the sex of parrots (FOCUS (BRL) 14:106-108 (1992)). Positive colonies are identified by autoradiography or a betascope, and identified on the control plates which are incubated without a nylon filter because of the similarity in pattern of application which is used. After identification the colonies are transferred into LB medium without agar, containing 50 ~g penicillin per ml, and incubated overnight at 37 C. The bacteria are collected by centrifugation and resuspended in LB + ampicillin containing 15% glycerol and stored at -80-C.
ExamDle II
L~ NATIoN OF THE SEX OF PARROTS WITH
THE AFRICAN GREY PARROT SEX SPECIFIC DNA OOl.~tl~
In carrying out a procedure in accordance with the present invention to determine the sex of a parrot, twenty-five (25) ~1 blood is removed from a wing vein or toe with a heparinized capillary and WO9~l02605 PCT~S94/08~
~16~29 l suspended in 0.5 ml of buffer A (a solution containing 0.05 M Na2EDTA and 0.15 M NaCl (pH 7.4)).
Of this suspension, 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. Since avian blood contains approximately 5 ~g DNA per ~l, this aqueous phase contains approximately 25 (5 x 5) ~g of DNA per 250 ~l, or l ~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 Na2EDTA), and the DNA is denatured (make single stranded) by the addition of 100 ~l of l M
NaOH. After five (5) minutes, the denatured DNA is neutralized with 200 ~l l M NaH2PO4 and 100 ~l of 1.5 M NaCl is added to a final volume of 500 ~l. 100 ~l of this solution (containing approximately l ~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. For hybridization, the filter with the bound denatured DNA is then preincub~ted for thirty minutes at 60-C with 0.2 ml ~095/02605 PCT~S94108023 - ~167~9S
per cm2 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. Subsequently, 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.
In the event the blood sample is taken from a blood feather, 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) ~f buffer A (see above) or any other stabilizing solution (Dulbecco's modified Eagles etc.). To this suspension is added 75-150 ~1 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 ~esulting cells as described above for blood W095102605 21~ 7 2 9 ~ PCT~S94/08C
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.
It should be understood that the procedure described is used to generate the results in Example III and as depicted in FIG. 3. It should also be understood that the procedures described herein are used to sexually type the parrots listed in Table I.
'~095/0~05 ~6~5. PCT~S94/08023 TABLE I
Species Female male - Blackmasked lovebird 8.3 0.3 Ring parrot 40.2 2.2 Bluefronted amazone 7.6 0.7 Double yellow headed amazone 8.4 o.7 Aymara parakeet 15.8 1.5 Crimsonwinged parrot 16.4 1.3 Blue and gold macaw 12.4 1.2 Scarlet macaw 14.7 O.g Severes macaw 13.8 1.4 Redfronted conure 15.8 1.3 Sun conure 17.2 1.7 Goffins cockatoo 30.3 O.9 Moluccan cockatoo 35.0 1.3 Sulphur-creasted cockatoo 33.7 1.1 Gang-gang cockatoo 30.2 0.8 White-tailed black cockatoo 38.3 0.7 Red-tailed black cockatoo 35.3 o.g Rosebreasted cockatoo 31.3 1.1 Chattering lory 13.5 1.3 Budgerigar 23.5 0.7 Cockatiel 34.5 0.6 Blue-headed pionus 12.4 0.9 Eastern rosella 25.6 0.4 Princess of Wales parakeet 24.3 0.5 Palm cockatoo 12.7 0.7 Alexandrine parakeet 20.6 1.3 Ring-necked parakeet 30.3 2.1 African grey parrot lOO.O 2.5 Pes~uet's parrot 8.5 0.3 Table I. Use of PARsexl for molecular sex determina-tion of selected psittacines. Values represent the average amount of PARSEXl present in DNA of 2 - 5 males and females of the species indicated, calculated as percentage of the amount present in the female African grey parrot. PARsexl refers to the DNA probe having the sequence shown in FIG. 1.
PC~ 9 4 1 0 8 ~ 2 3 -;~93~ IPEA/US ~2AUG 199 ~ ple IT~
RF~JT.'rS
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 Psittacus , namely, the conure, the cockatoo, the lory, the mazone 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. In FIG. 3, different amounts of DNA of males and females of the species indicated are immobilized on a nylon filter and hydribized with the p32-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 speci~ic DNA
component is structurally conserved between the different species and can be used for the determination of all species.
~0~
VOg5/0~05 - 27 21 6 72 9 5 PCT~S94/080~
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: de Kloet, Siwo R.
(ii) TITLE OF INv~NllON: Sex-Specific DNA Probe For Parrots, Methods And Kits (iii) NUMBER OF SEQUENCES: 44 (iv) CORRESPONDENCE ~nn~.c,~
(A) ADDRESSEE: Ruden, Barnett, McClosky, Smith, Schuster &
Russell, P.A.
(B) STREET: 200 East Broward Boulevard (C) CITY: Fort Lauderdale (D) STATE: FL
(E) COUNTRY: USA
(F) ZIP: 33301 (v) CGI.~ul~:K ~An~RT.~ FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/093,198 (B) FILING DATE: 15-JUL-1993 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT lN~O~IATION:
(A) NAME: Manso, Peter J.
(B) REGISTRATION NUMBER: 32,264 (C) REFERENCE/DOCKET NUMBER: FL20979-34 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 305-527-2498 (B) TELEFAX: 305-764-4996 (2) INFORMATION FOR SEQ ID NO:1:
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GTTTTCGTCC ATTCCTAACC ACATTAAAGC CTAlllllCA CCCATTTCCA ACCAATTTTA 60 wo gs/o~os ~ 16 ~ 2 9 ~ - 28 - PCT~S94/080' rTGGTTGCTT GTAAATGATT TTTGGAGTTT TCTAACCCCT TTTGAGCCAT TTTTTCTGTT 180 TCTAACCCAT TTTTTCAACA GTTCTAGCTC GGTTTAAGTA G~ GCTT TTTTCTAACC 240 CATTGATCCC ATGACTAATC AGTTTTAAGC C~lllllGlC CATTTCTGAC CCATCTTTGC 300 CCAGCTCTAG ~lll~lllAA GCC~lllllC TCCATTTCTA ACCCGTTTCT AGCCCATTCC 360 AlllllCACC TCTTCTGACT CGCTTGAAGA C~lllllGCC 460 (2) INFORMATION FOR SEQ ID NO:2:
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C~ GCCG 10 W095/0~05 40 PCT~S94/08C
2l6~ ~9~
The present invention may, of course, be carried out in other specific ways than those herein set forth without departing from the spirit and essential characteristics of the invention. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive and all changes coming within the spirit and scope of the appended claims are intended to be embraced herein.
Having described my invention, I claim:
sulphur-crested cockatoo (Cacatua galerita); 4 blue-fronted amazon (Amazona aestiva); 5 = blue and gold macaw (Ara ararauna); 6 = African grey parrot (Pisttacus erithacus).
Detailed Descri~tion By way of illustrating and providing a more complete appreciation of the present invention and many of the att~n~t advantages thereof, the following detailed description is provided concerning the novel nucleotide sequences, methods and kits.
The 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 MspI, in the vector pGEM3Z+ and the host Eschericia coli 31/17.
The 461 base pair repeat is derived and isolated from -a base pair repeat of about 570 base pairs and is made up of 43 subfragments in which oligo dT
fragments of 2-6 thymine residues are repeated at an average distance of lO.Sbp. More specifically, the 2-6 long oligo T fragments are part of 43 subfragments of a minimum length of about 4 nts and a maximum ~ength of about 20 nts, and an average length ~095/0~05 PCT~S94/080~
216729~
of about 10.5 nts which together form the 461 nt long fragment which is cloned. This latter fragment is again tandemly repeated 12,000 times. 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.
FOCUS (BRL) 14: 106-108 (1992). 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.
W095/0~05 ~6~ 295 PCT~S94/08 Utilizing this procedure with the restriction enzyme MspI, 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 MspI 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 circular~zation experiments. Curved DNA exhibits an vo 95~0260s 21 6 7295 PCT~S94/080~
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. Hence, 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 F the DNA probes of the present invention may ~167~?9S- PCT~JS 94/0~423 IP~ JS 3 :AUG 1995 be modified by adding, deleting and/or substitutingnucleotides 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. 1, which is complimentary to a DNA~segment on a W chromosome which is characteristic for a female parrot of the Psittacus genus is contemplated by the present invention. Examples of active nucleotide fragments in accordance with the present invention are those fragments derived by, for example, digesting the 461 base pair fragment with t.~ el,zyme SAU3Al at a GATC
site using diges~ion t.~hn.i ~ known to those skilled in the art. When` ~h~ 461 base pair fragment is digested with the enzyme SAU3Al, the 461 base pair 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 co~e~onding to bases 1-248 and 249-461 in the 5'-3' sequence and _~9~J
2167,~95 - PC~IS 94/08 023 IPEAJUS ~}~AUG ~
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 gualified personnel interested in sex-typing parrots~ of the Psittacus genus. A
typical kit in accordance with this invention includes blood sample stabilizing Solution A;
lo proteinase-K and sodium sarcosylate Solutions, or a commercially available fast DNA isolation kit; D~A
denaturing and neutralizing Solutions; nylon membranes; biotin-labelled sex specific DNA or RNA
probe; hybridization Solution ~: 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 pres~nt invention is standard laboratory e~uipm~t.
According to the prese~lt invention, 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.
In carrying out the sex typing of parrots in accordance with the present invention, blood samples, approximately 25 to 100 ~1, 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 ,~D ~r Yo 95,~.605 ~ 1 6 7 2 9S PCT~S94/08023 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. ~When chloroform extraction is used, the chloroform should be removed by for example centrifugation.~ Accord~ 1 of the 1:1 diluted blood sample ~(containing on average 50 mgs of DNA) is diluted with 90 ~1 of Solution A and incubated for one hour at about 65-C with proteinase K (2 ~gs per 100 ~1) 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 NaH2PO4. Samples containing approximately 1 mg of denatured parrot DNA
VOg5/02605 PCT~S94/080~
~` 2~1 6729~
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.
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, O.l M Tris-HCl buffer pH
7.8, 0.05 M Na2EDTA, 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. 1, is tagged by enzymatic or nonenzymatic procedures with radioactive p32 or nonradioactive biotin or fluorescent groups like fluoresceine or rho~mine using technologies available to these in the labeling art. It should be understood that the DNA probe may also be tagged with fluorescent dyes, such as fluoreseine or rhodamine using techni~ues known to those versed in this art. In the present example the DNA probe is tagged by, e.g., labelling with biodUTP
using nick translation or random primer extension.
As to an RNA probe, it is labelled with, e.g., bioUTP
using T~ RNA polymerase (pGEM3Z+ has a T7 RNA
WO 9S/02C05 ; Z l 6 ~ PCT~S94/080 polymerase promoter site). The tagged, denatured DNA
probe is then incubated for approximately 60 - 65-C
and at a concentration of about 25 ng per ml of Solution B with the nylon filters containing the parrot DNA. Subsequent to incubation, the filters are washed first at room temperature with 1 x SSC (=
0.15 M NaCl, 0.015 M sodium citrate pH. 7.0, Solution C) (five times for about five minutes with about lO0 ml) and subse~uently one time for about two minutes at about 55-C with approximately 100 ml 1 x SSC
containing about 0.1% sodium lauryl sulphate (Solution D). Binding of radioactively labelled probe is visualized and quantified by autoradiography and/or a betascope. In this Example, bound probes labelled nonradioactively with biotin are visualized through a commercially available detection process which involves a specific color reaction or chemiluminescence. The specific colour reaction involves, e.g., the binding of a streptavidin or avidin phosphatase conjugate to the biotin residue -(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 ~hosphate by the phosphatase. Examples of VOg5/0~05 t 6 7~9 ~ PCT~Sg4tO8023 such 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.
The present invention will now be further illustrated with reference to the following Examples.
ExamPle I
SEX ~r.~ lC DNA F'177'r~F~IT
Female african grey parrot DNA (10 ug) is digested in 100 ~1 of a buffer (containing 6 mM
MgC12, 6 mM Tris-HCl buffer pH 7.5, 50 mM NaCl) with 20 units of the restriction endonuclease Mspl for 3 hrs at 37- C. For identification purposes of the sex specific DNA fragment, 10 ~1 of the digest is fractionnated by electrophoresis through a 1% agarose gel in TPE buffer (TPE = 0.08 M Tris-phosphate pH
7.5, 0.008 M EDTA). A southern blot of the gel is made and probed with a genomic DNA probe made from 25 ng radioactively (p32) labelled female DNA [labelled by random primer extension, Feinberg, A.P. et al.:
Anal. Biochem., 132:6-13 (1983), preannealed to 100 2 5 ~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
W095l02605 PCT~S94/080 2 g ~
(1992) Molecular determination of the sex of parrots (FOCUS (BRL) 14:106-108 (1992)).
After identification of the sex specific DNA fragment by autoradiography or with a betascope, 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 ACC1 site of the plasmid pGEM3Z+ and the ligation product is used to transform E. coli 71/18 using the CaC12 procedure (Maniatis, T. et al.
(1982) "Molecular cloning; a laboratory manual."
Cold Spring Harbour Laboratory. Cold Spring Harbour, NY, 2) probe labelling with biotin:). The transformed bacteria are spread on 20 ml agar plates containing Luria bertani (L8) medium (LB = 1%
bacto=tryptone, 1% NaCl, 0.5% bacto yeast extract pH
7.4) containing per ml 50 ~gs of penicillin, 150 ~gs bromo-idnolyl-beta-galactoside and 75 ~g isopropyl beta thiogalactoside. After growth vernight at 37-C, the white colonies (containing plasmids with inserts) are selected and transferred to fresh FL plates covered ~ith a Nylon filter. Control plates without ~095t02605 1 6 729 ~ PCT~S94/08023 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. Cold Spring Harbour, NY, 2) probe labelling with biotin:) using the same genomic sex lo specific DNA probe as is used initially for determination of the sex of parrots (FOCUS (BRL) 14:106-108 (1992)). Positive colonies are identified by autoradiography or a betascope, and identified on the control plates which are incubated without a nylon filter because of the similarity in pattern of application which is used. After identification the colonies are transferred into LB medium without agar, containing 50 ~g penicillin per ml, and incubated overnight at 37 C. The bacteria are collected by centrifugation and resuspended in LB + ampicillin containing 15% glycerol and stored at -80-C.
ExamDle II
L~ NATIoN OF THE SEX OF PARROTS WITH
THE AFRICAN GREY PARROT SEX SPECIFIC DNA OOl.~tl~
In carrying out a procedure in accordance with the present invention to determine the sex of a parrot, twenty-five (25) ~1 blood is removed from a wing vein or toe with a heparinized capillary and WO9~l02605 PCT~S94/08~
~16~29 l suspended in 0.5 ml of buffer A (a solution containing 0.05 M Na2EDTA and 0.15 M NaCl (pH 7.4)).
Of this suspension, 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. Since avian blood contains approximately 5 ~g DNA per ~l, this aqueous phase contains approximately 25 (5 x 5) ~g of DNA per 250 ~l, or l ~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 Na2EDTA), and the DNA is denatured (make single stranded) by the addition of 100 ~l of l M
NaOH. After five (5) minutes, the denatured DNA is neutralized with 200 ~l l M NaH2PO4 and 100 ~l of 1.5 M NaCl is added to a final volume of 500 ~l. 100 ~l of this solution (containing approximately l ~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. For hybridization, the filter with the bound denatured DNA is then preincub~ted for thirty minutes at 60-C with 0.2 ml ~095/02605 PCT~S94108023 - ~167~9S
per cm2 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. Subsequently, 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.
In the event the blood sample is taken from a blood feather, 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) ~f buffer A (see above) or any other stabilizing solution (Dulbecco's modified Eagles etc.). To this suspension is added 75-150 ~1 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 ~esulting cells as described above for blood W095102605 21~ 7 2 9 ~ PCT~S94/08C
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.
It should be understood that the procedure described is used to generate the results in Example III and as depicted in FIG. 3. It should also be understood that the procedures described herein are used to sexually type the parrots listed in Table I.
'~095/0~05 ~6~5. PCT~S94/08023 TABLE I
Species Female male - Blackmasked lovebird 8.3 0.3 Ring parrot 40.2 2.2 Bluefronted amazone 7.6 0.7 Double yellow headed amazone 8.4 o.7 Aymara parakeet 15.8 1.5 Crimsonwinged parrot 16.4 1.3 Blue and gold macaw 12.4 1.2 Scarlet macaw 14.7 O.g Severes macaw 13.8 1.4 Redfronted conure 15.8 1.3 Sun conure 17.2 1.7 Goffins cockatoo 30.3 O.9 Moluccan cockatoo 35.0 1.3 Sulphur-creasted cockatoo 33.7 1.1 Gang-gang cockatoo 30.2 0.8 White-tailed black cockatoo 38.3 0.7 Red-tailed black cockatoo 35.3 o.g Rosebreasted cockatoo 31.3 1.1 Chattering lory 13.5 1.3 Budgerigar 23.5 0.7 Cockatiel 34.5 0.6 Blue-headed pionus 12.4 0.9 Eastern rosella 25.6 0.4 Princess of Wales parakeet 24.3 0.5 Palm cockatoo 12.7 0.7 Alexandrine parakeet 20.6 1.3 Ring-necked parakeet 30.3 2.1 African grey parrot lOO.O 2.5 Pes~uet's parrot 8.5 0.3 Table I. Use of PARsexl for molecular sex determina-tion of selected psittacines. Values represent the average amount of PARSEXl present in DNA of 2 - 5 males and females of the species indicated, calculated as percentage of the amount present in the female African grey parrot. PARsexl refers to the DNA probe having the sequence shown in FIG. 1.
PC~ 9 4 1 0 8 ~ 2 3 -;~93~ IPEA/US ~2AUG 199 ~ ple IT~
RF~JT.'rS
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 Psittacus , namely, the conure, the cockatoo, the lory, the mazone 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. In FIG. 3, different amounts of DNA of males and females of the species indicated are immobilized on a nylon filter and hydribized with the p32-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 speci~ic DNA
component is structurally conserved between the different species and can be used for the determination of all species.
~0~
VOg5/0~05 - 27 21 6 72 9 5 PCT~S94/080~
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: de Kloet, Siwo R.
(ii) TITLE OF INv~NllON: Sex-Specific DNA Probe For Parrots, Methods And Kits (iii) NUMBER OF SEQUENCES: 44 (iv) CORRESPONDENCE ~nn~.c,~
(A) ADDRESSEE: Ruden, Barnett, McClosky, Smith, Schuster &
Russell, P.A.
(B) STREET: 200 East Broward Boulevard (C) CITY: Fort Lauderdale (D) STATE: FL
(E) COUNTRY: USA
(F) ZIP: 33301 (v) CGI.~ul~:K ~An~RT.~ FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/093,198 (B) FILING DATE: 15-JUL-1993 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT lN~O~IATION:
(A) NAME: Manso, Peter J.
(B) REGISTRATION NUMBER: 32,264 (C) REFERENCE/DOCKET NUMBER: FL20979-34 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 305-527-2498 (B) TELEFAX: 305-764-4996 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 460 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GTTTTCGTCC ATTCCTAACC ACATTAAAGC CTAlllllCA CCCATTTCCA ACCAATTTTA 60 wo gs/o~os ~ 16 ~ 2 9 ~ - 28 - PCT~S94/080' rTGGTTGCTT GTAAATGATT TTTGGAGTTT TCTAACCCCT TTTGAGCCAT TTTTTCTGTT 180 TCTAACCCAT TTTTTCAACA GTTCTAGCTC GGTTTAAGTA G~ GCTT TTTTCTAACC 240 CATTGATCCC ATGACTAATC AGTTTTAAGC C~lllllGlC CATTTCTGAC CCATCTTTGC 300 CCAGCTCTAG ~lll~lllAA GCC~lllllC TCCATTTCTA ACCCGTTTCT AGCCCATTCC 360 AlllllCACC TCTTCTGACT CGCTTGAAGA C~lllllGCC 460 (2) INFORMATION FOR SEQ ID NO:2:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lO base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
C~ GCCG 10 W095/0~05 40 PCT~S94/08C
2l6~ ~9~
The present invention may, of course, be carried out in other specific ways than those herein set forth without departing from the spirit and essential characteristics of the invention. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive and all changes coming within the spirit and scope of the appended claims are intended to be embraced herein.
Having described my invention, I claim:
Claims
(2) An oligonucleotide probe as recited in claim 1, said probe further including a labeled moiety so that, when said labeled probe is hybridized to the DNA
segment, the DNA segment on the W chromosome can be detected.
(3) An oligonucleotide probe as recited in claim 2, said labeled moiety being a fluorophore.
(4) An oligonucleotide probe as recited in claim 3, said fluorophore being selected from a group consisting of a fluoroscein, a rhodamine or any other fluorophore.
(5) An oligonucleotide probe as recited in claim 2, said labeled moiety being selected from a group consisting of biotin and a radioisotope.
(6) An oligonucleotide probe as recited in claim 1, said probe having a periodicity of groups of about 2 to about 6 thymine or adenine residues, said groups being repeated on average of about 10.5 nucleotides.
(7) An oligonucleotide probe as recited in claim 1, said oligonucleotide probe further having a curved DNA element.
(8) An oligonucleotide probe as recited in claim 1, said oligonucleotide probe having a nucleotide sequence selected from a group having the following 5'-3' sequence or 3'-5' sequence or an equivalent sequence or an effective segment thereof:
(9) An oligonucleotide probe as recited in claim 1, said probe being a DNA probe.
(10) An oligonucleotide probe as recited in claim 1, said probe being a RNA probe.
(11) A method of determining the sex of a parrot of the Psittacus genus, said method comprising:
hybridizing the labeled probe of claim 2 to the female chromosome DNA of the animal; and detecting the labeled probe hybridized to the DNA segment on the female chromosome of the parrot to identify the female chromosome of the parrot.
(12) A method as recited in claim 11, said detecting step comprises:
visualizing the labeled probe hybridized to the DNA segment.
(13) A method as recited in claim 11, the labeled probe being a labeled DNA probe.
(14) A method as recited in claim 11, the labeled probe being a labeled RNA probe.
(15) A kit for determining the sex of a parrot of the Psittacus genus, said kit comprising:
a.) an oligonucleotide probe of claim 1; and b.) components for carrying out an assay to determine the sex of the parrot.
(16) A kit as recited in claim 15, said oligonucleotide probe being a DNA probe.
(17) A kit as recited in claim 15, said oligonucleotide probe being a RNA probe.
(18) A method of determining the sex of a parrot of the Psittacus genus, said method comprising:
exposing the oligonucleotide probe of claim
1 to a DNA segment of the W chromosome obtained from the parrot; and determining if the oligonucleotide probe is hybridized to the DNA segment, so that in the event the oligonucleotide probe is hybridized to the DNA segment the parrot is identified as a female and in the event the oligonucleotide probe is not hybridized to the DNA
segment the parrot is identified as a male.
(19) A method as recited in claim 18, the oligonucleotide probe further including a labeled moiety, and said determining step comprises:
visualizing the labeled oligonucleotide probe when hybridized to the DNA segment to identify the parrot as a female.
segment the parrot is identified as a male.
(19) A method as recited in claim 18, the oligonucleotide probe further including a labeled moiety, and said determining step comprises:
visualizing the labeled oligonucleotide probe when hybridized to the DNA segment to identify the parrot as a female.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US9319893A | 1993-07-15 | 1993-07-15 | |
| US093,198 | 1993-07-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2167295A1 true CA2167295A1 (en) | 1995-01-26 |
Family
ID=22237701
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002167295A Abandoned CA2167295A1 (en) | 1993-07-15 | 1994-07-15 | Sex-specific dna probe for parrots, methods and kits |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0708782A4 (en) |
| AU (1) | AU7364594A (en) |
| CA (1) | CA2167295A1 (en) |
| WO (1) | WO1995002605A1 (en) |
Families Citing this family (1)
| 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 |
-
1994
- 1994-07-15 WO PCT/US1994/008023 patent/WO1995002605A1/en not_active Application Discontinuation
- 1994-07-15 AU AU73645/94A patent/AU7364594A/en not_active Abandoned
- 1994-07-15 CA CA002167295A patent/CA2167295A1/en not_active Abandoned
- 1994-07-15 EP EP94922588A patent/EP0708782A4/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| EP0708782A1 (en) | 1996-05-01 |
| AU7364594A (en) | 1995-02-13 |
| EP0708782A4 (en) | 1997-01-02 |
| WO1995002605A1 (en) | 1995-01-26 |
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