Title of Invention


Abstract The present invention relates to oligonucleotide probe capable of identifying only Y-chromosome specific DNA in buffalo. Further, the present invention provides a process for preparing a novel oligonucleotide probe.
The present invention relates to oligonucleotide probe capable of identifying only Y-chromosome specific DNA in buffalo. More specifically, the present invention provides a process for preparing a novel oligonucleotide probe.
The capacity of determine the sex of an embryo or a fetus is becoming increasingly advantageous, particularly in the light of advances in the area of reproductive biology such as embryo transfer. In the dairy and beef cattle industry alone, some 50,000 embryo transplants were reported to have been carried out in 1985. Given the predisposition of the dairy industry to female progeny, it would be most advantageous if embryos could be routinely sexed prior to transfer into a maternal host. The availability of sexed embryos would allow dairy producers to select replacement progeny for their stock from embryos which possessed desirable traits, such as increased milk production and mothering ability. Similarly in the sheep and goat industries, the availability of sexed embryos would enable producers to select the most desirable progeny for their stock.
The ability to determine the sex of an embryo or foetus in vitro is also important. In "conventional" pregnancies which do not involve embryo transfer, but rather arise via artificial insemination or natural insemination, the early determination of sex of an embryo or foetus would allow a producer to terminate a pregnancy if an embryo or foetus of the desired sex was not obtained.
The primary sex of a mammal is determined by the presence or absence of the entire Y-chromosome or a functional portion thereof. Gene(s) present on the Y-chromosome are responsible for the formation of the testis, and the development of the male phenotype. The primary sex of an individual mammal is, therefore, dependent upon whether or not its genome contains certain DNA sequences, specifically those sequences comprising that part of the Y-chromosome which encode gene(s) responsible for testis determination.
In other words, in mammals, sex determination is genetically controlled by the 'Y'-chromosome. An embryo that inherits a Y-chromosome develops into a male, whereas an embryo lacking a Y-chromosome develops into a female. Therefore, sex can be determined by detection of Y-chromosome specific DNA sequences. DNA sequences specific to the bovine (cattle) Y-chromosome have been cloned by various groups and successfully used for sex determiantion of cattle embryos using PCR (Leonard, 1987 ; Bondoli, 1989). However, it is unknown whether these sequences are capable of being used for sex determination in buffalo.
Therefore, the sex or presumptive sex of an individual mammal can be determined by analysis for Y-chromosome specific genes in the DNA of the animal. Alternatively, sex can be determined by analysis for unrelated but genetically linked sequences that are associated specifically with the Y-chromosome. In order to minimise possible errors due to infrequent genetic recombination events, such analysis are best made for sequences which are linked closely to the testis-determining gene(s).
In short, in mammals, sex determination is genetically controlled by the Y-chromosome; embryos having the Y-chromosome develop testes and become males regardless of the number of X-chromosomes.. The dominant function of Y-
chromosome is therefore, to induce bipotential undifferentiated gonads to become testis and in its absence ovaries develop (Gordon and Ruddle, 1981). Recently many groups have isolated several Y-chromosome specific sequences from mouse (Lamar and Palmer, 1984), human (Goodfellow et al., 1985), cattle (Matthews and Reed, 1991), pig (McGraw et al., 1988) and sheep (Herr et al., 1990). Amongst, livestock species about 10 male-specific DNA sequences in cattle have been cloned, primarily for the purpose of sexing of embryos (Miller, 1991). In all species examined a large part of Y-specific DNA appears to consists of repeated sequences, with a copy number ranging from 20 to 60000 (Kashi et al., 1990; Ferret et al., 1990).
Although, Y-chromosome specific DNA's could be used as a probe to sex preimplantation embryos (Bondioli et al., 1989), the techniques necessitat autoradiography and is time consuming. The polymerase chain reaction, on the otherhand, is rapid, reliable and simple method. PCR has been successfully used to identify the sex of embryos before transfer (Handyside et al., 1989; Appa Rao et al., 1994). Since, predetermination of sex of offspring of agriculturally important species has been an objective of animal breeders, many Y-specific sequences have been isolated and characterized. One such sequence is designated BRY.l, a bovine repeat and Y-associated. BRY.l is quantitatively polymorphic and is present in multiple copies in males. Further, it is also documented that BRY.l has a Y-associated homologue in sheep and goat (Matthews and Reed, 1992).
Buffaloes (Bubalus bubalis) belongs to family Bovidae, which constitute the backbone of the dairy industry in India. Therefore, there is a need here to improve and to produce more buffaloes faster employing sex determination in conjunction with embryo transfer technology. It would assist embryo transfer technology in selecting out elite buffalo females and utilizing them in creating a
sizeable buffalo male population for breeding purposes. Sex determination of embryo by routine use of this method can help to create a nucleus embryo repository in the country. This will allow farmers to choose the embryo with predetermined sex of their choice from repository for the sake of maximum economic returns. This will ensure that male embryos suitably go for breeding centre for the production of large number of genetically superior progency tested bulls to support A.I. network.
Recently, a homologue of BRY.l from buffalo has been amplified by PCR in sex determination of buffalo embryos (Appa rao et al., 1993), suggesting that BRY.l is conserved in buffalo also. Preselection of sex of offspring of such species would have tremendous impact on economy as they are rugged for the harsh tropical climate.
Number of investigators have identified DNA sequences which hybridize preferentially or exclusively to male DNA. These DNA sequences have not been functionally characterized. Furthermore, it is unknown whether these sequences are capable of hybridization to non-human species (Matthews and Reed, 1991; Miller, 199l,Lamar and Palmer, 1984).
Australian patent application No. 59561/86 discloses bovine DNA probes which hybridize preferentially to male DNA. These DNA sequences are stated to be useful as hybridization probes for sexing hi embryos and fetuses. This DNA probe is species specific i.e. bovine and hybridize to DNA from other ruminant animals such as Buffaloes least efficiently.
Detailed description of the invention
The present invention arises from the discovery of a Y-chromosome specific DNA sequence which is specific to buffalo.
One embodiment of the invention provides an isolated DNA sequence which is male specific and shown herebelow.
(Sequence Removed)
Nucleotide sequence of male specific repetitive DNA fragment from Buffalo "BuRY.I" (GenBank Accession # X93551), amplified using BRY.I primers. The external primers used for primary amplification are underlined. The sequencec used to construct the nested primers are also shown in italics with underlining.
The present isolated nucleic acid sequence comprising single or double strands of the 301 base pairs of BYS.l, which is capable of detecting Y chromosome sequence in buffalo, cattle, sheep and goat. The said isolated nucleotides sequences of the invention comprising ribonucleiotides.
Another embodiment of the invention relates to a method for sex determination of a tissue from the said animals, which comprises isolating DNA from a tissue or cell sample obtained from an individual buffalo, cattle, sheep or goat; immobilizing said isolated DNA with a single or double stranded nucleotide sequence as described above under conditions whereby the said single stranded nucleotide sequence selectively hybridizes with the Y chromosome if said chromosome is present in said tissue or cell sample; washing unbound nucleic

acids from the support matrix and then detecting the binding of said single stranded nucleic acid to said isolated immobilized DNA whereby binding indicates the presence of Y chromosomes and the absence of binding indicates the absence of Y chromosome.
The preferred tissue or cell sample used in the above process is obtained from an embryo, a fetus or from sperm.
Yet another embodiment of the invention relates to an isolated nucleotide sequences capable of detecting Y-chromosome sequences of buffalo, cattle, sheep and goat; said nucleotides having high degree of homology with respect to DNA sequence shown earlier, said degree of homology being defined as divergence by no more than 22% of the nucleotide sequences whereby said isolated nucleotide sequence having a high degree of homology is capable of selectively hybridizing to the nucleotide sequence.
Yet another embodiment of the invention provides a method for determining the presence or absence of a Y-chromosome in buffalo, cattle, sheep and goats which comprises hybridizing fixed cells or metaphase chromosome spreads from an individual buffalo, cattle, sheep or goat with the single or double stranded nucleotide sequences.
Still another embodiment of the invention relates to a method for detecting the presence of sex chromosome constitution of ruminants including buffalo, cattle, sheep and goat which comprises isolating DNA from tissue or cell sample obtained from an individual buffalo, cattle, sheep or goat; denaturing the isolated DNA to separate respective coding and non-coding strands; annealing the denatured DNA with a nucleotides sequence; incubating the annealed DNA with DNA

polymerase to extend the polynucleiotide through the BYS.l DNA sequence if present hi the tissue or cell sample; repeating this sequence as many tunes as desired to amplify levels of BYS.l and subsequently detecting BYS.l DNA sequence in the amplified sample whereby the presence of said BYS.l DNA sequences indicates the presence of the Y-chromosome and absence of BYS.l DNA sequences indicates the absence of said Y-chromosome.
According to still another aspect of the present invention, there is provided a nucleic acid isolate capable of hybridizing only to Y-chromosome specific DNA species of buffalo but also hybridize cattle, sheep and goats.
The nucleic acid isolate corresponds to all or part of a DNA sequence found on the Y chromosome of buffalo animals referred to herein after BYS.l.
The novel BYS.l sequence is shown herebelow:
(Sequence Removed)
Nuclcotide sequence of male specific repetitive DNA fragment from Buffalo "BuRY.I" (GenBank Accession # X93551), amplified using BRY.I primers. The external primers used for primary amplification are underlined. The sequencec used to construct the nested primers are also shown in italics with underlining.
This sequence comprise 301 nucleotides and shows 88% homology with cattle sequences. Further , in order to obtain specificity for buffalo, the nested primers which contained contiguous portion of 20 or more nucleotides of BYS.l on the basis of buffalo sequence were designed and used for sex determination of buffalo embryos. The resulting using nested primers indicated that sex can be determined to 100% accuracy.
Brief description of the drawings
Fig:l Accuracy of primary PCR for identifying workability of buffalo specific primers Bu RYN.I in DNA of different species such as cattle, Sheep and Goat. Amplification of Buffalo DNA is more than other species.
Fig-,2. Accuracy of primary PCR for sexing embryos. Each embryonic sample was divided into two and amplified with different primers. Top panel (a: sample 1-10) amplified with BRY.I primers in Primary PCR and bottom panel (b) represents samples amplified with BuRYN.I primers in nested PCR. Lanes 1,2,4,7,9,10 were assigned as male embryos for presenting a 301 (a) and 164 (b) bp product and lanes 3,5,6,8 were assigned as females as no amplification is observed. Lane m: 0X174 DNA/Hae III as molecular size marker.
Fig:3 Multiplex PCR for sexing of buffalo IVF embryos using BuRYN.I primers and ZFX/ZFY primers. ZFX/ZFY loci specific primers indicates gender-neutral signals (445 bp) and thus, acts as a positive control and BuRYN.I primers shows male specific signals (164 bp). Lanes 1,3,4,6,9 are male embryos and lanes 2,5,7,8,10,11 are female embryos. Lane M: 0X174 DNA digested with Hae III as molecular size marker.
DNA Extraction
Genomic DNA was extracted from venous blood lymphocytes of male and female buffalo, as described by Appa Rao et al (1994). Briefly, after blood cells were lysed, the cell pellet was incubated in lysis buffer (70 mM NaCl, 20 mM EDTA pH: 8.0, 0.5% (w/v) sodium dodecyl sulfate (SDS) and proteinase K (200 ug/rnl) and incubated at 37°C for overnight. The lysate was subsequently extracted twice with a phenol-chloroform-isoarnylalcohol mixture and DNA was precipitated with absolute ethanol at -20°C.
Amplification of BRY.I Homologue Sequence from Buffalo Genome by PCR
The PCR was carried out in a total volume of 25 ul containing approximately 50 ng of genomic DNA using BRY.I specific primers. Reaction mixture contains genomic DNA, 20 pM of each primer, 200 uM of each dNTPs, 2.0 mM MgC12, IX PCR buffer (10 mM Tris.HCl pH:8.3, 50 mM KC1) and 1.25 U of Taq DNA polymerase (GEBCO, BRL). After centrifugation briefly, the reaction mixtures were overlaid with two drops of light mineral oil before amplification in order to prevent evaporation. The amplifications were carried out hi a programmed Thermal Cycler (The Perkin-Elmer Corp., Norwalk, CT, USA). Initial denaturation was at 94°C for 5 mm, followed by 40 cycles of 94°C denaturation (30 sec), .60°C annealing (45 sec) and 72°C extension (45 sec), with a final extension at 72°C for 5 mm. Ten to 15 ul of amplified DNA was electrophoresefit for 45 min at 100 V on 1.8% agarose gel, stained with 0.5 ug/ml of ethidium bromide and visualized with UV transilluminator.
Southern blot analysis
Ten ul of PCR products amplified using BRY.I primers on cattle, buffalo, sheep
and goat genomic DNA were resolved on 1.8% agarose gel. DNA's were
denatured with an alkaline solution, neutralized with Tris buffer (pH:8.0) and then transferred onto a nylon membrane (Hybond-NQTM, Amersham) by capillary blotting following standard procedure (Southern, 1975). A BRY.I homologue, designated as Buffalo Y-specific repetitive sequence (BuRY.I) amplified from buffalo genomic DNA was gel eluted and labeled with (D SYMBOL 97 \f "Symbol" D-32p) dCTP by random priming (Vogelstein, 1984) and used as a probe. The blots were hybridized for 18 h at 60DSYMBOL 176 \f"Symbol"DC in a mixture containing 5X SSC, 5X Denhardts solution, 0.5% SDS and 100 ug/ml of sonicated denatured salmon sperm DNA. After hybridization, the membranes were washed for 15 min twice at room temperature in 2XSSC plus 0.1% SDS followed by hot washes with 0.2XSSC plus 0.5% SDS. Autoradiography was performed with Kodak XAR-5 film exposed with Cronex lightning plus intensifying screens at -70°C for 3-24 h.
Cloning and Sequencing of BuRY.I Sequence and Primer design
BuRY.I sequence was excised from agarose gels, reamplified and ligated into pGEM 4Z+ vector and transformed in E.coli strain JM109. Recombinant clones were amplified for overnight in LB medium and plasmid DNA was extracted following standard procedure (Birnboim and Doly, 1979). The plasmid DNA was purified by lithium chloride-polyethelene glycol, ethanol precipitated and redissolved in sterile distilled water. The plasmid DNA was then sequenced from both strands with Sanger's dideoxy chain termination sequencing method (Sequenase version 2.0 from USA, Cleveland, Ohio) using forward (T7) and reverse (SP6) primers. The 35s-labelled product was separated on a 5% acrylamide, 600x200x0.4 mm sequencing gel at 2000 volts. The dried gel was autoradiographed overnight and developed. Nucleotide sequence was analyzed using DNAsis Software package. Nested PCR primers were designed to produce BuRY.I-specific products. Primers locations were chosen to provide a difference
with BRY.I sequence. Additional mismatches were also incorporated in nested primers to ensure BuRY.I-specific amplification, as shown herebelow:
(Sequence Removed)
Nucleotide sequence of male specific repetitive DN/t fragment from Buffalo "BuRY.I" (Genbank Accession # X9355I, Appa Rao, K.B.C and Totey , S.M, deposited 28th November, 1995), amplified using BRY.I primers. The external primers used for primary amplification are underlined. The sequence used to construct the nested primers are also shown in italics with underlining.
In-Vitro embryo Production
In-vitro fertilized (IVF) embryos were produced by the method described elsewhere (Totey et al., 1992). Briefly, buffalo ovaries were obtained at a slaughterhouse and kept in a thermos with a normal saline and were transported to the laboratory at 25°C within 1-2 h of slaughter. Cumulus-oocyte-complexes (COCs) were aspirated, using a sterile disposable 18-g needle fixed to 10-ml plastic syringe, from antral follicles (2-6 mm in diameter). COCs were recovered
from the follicular fluid aspirates after centrifugation for 3 min at 500 rpm. COCs were subsequently washed two times with 4-(2-hydroxyethyl)-piperazine ethane sulfonic acid (HEPES) buffered tyrode medium (TL-HEPES). The COCs were matured in (10 per 100-ul drop) a maturation medium consisting of Ham's F-10 supplemented with 10% heat-inactivated fetal calf serum (PCS) and gonadotrophins (0.5 ug/ml oFSH and 5 ug/ml of oLH, NIDDK and 1 ug/ml of B-estradiol). All COCs were placed in petri dishes covered with mineral oil and incubated at 39°C in 5% CO2,95% humidity for 24 hours.
Frozen-thawed semen, derived from a single bull and tested for use in IVF, was washed using a discontinuous density percoll gradient. Matured oocytes were placed in 50-ul drops of fertilization medium and insemination with sperm at a concentration of 2x106 sperms/ml, previously treated with 10 ug/ml heparin for capacitation. After 20 h of co-incubation with live-motile spermatozoa at 39°C in 5% C02 and 95% humidity, presumptive zygotes washed in (TL-HEPES) and then co-cultured on buffalo oviductal epithelial cell monolayer in TC-199 medium supplemented with 10% PCS for further development. Embryos were evaluated morphologically for the development of morulae and blastocyst on day 6-8 of co-culture. Good quality morulae/blastocyst were used for sex determination.
Oligonucleotide Primers
Two pairs of Oligonucleotide primers, one for BRY.I (5'-
3'), a bovine Y-chromosome specific repetitive sequence (Reed et al., 1988) and
other for ZFX/ZFY loci specific primers (51-
GCACTTCTTTGGTATCTGAGAAAGT-31), designed by choosing sequences that conserved between the human and mouse ZFX/ZFY genes (Aasen and
Medrano, 1990) were used in the study apart from nested primers. The ZFX/ZFY loci was found to be conserved in all placenta! mammals and located on X and Y chromosome (Page et al., 1987) and thereby chosen for control primers. Simultaneously, nested PCR primers constructed based on BuRY.I sequence was designated as BuRYN.I (S'-CGTGGTGGGTGACCCCACAGCCCC-S1 and 5'-ACAGGTGCTTATGCTGCAGTGCTG-3') and located within the fragment amplified by corresponding outer pair of primers. All these primers were chemically synthesized by Rama Biotechnologies Pvt. Limited (India). Both BRY.I and ZFX/ZFY loci specific primers yields a 301 bp and 445 bp fragment respectively, while the nested primer pair, BuRYN.I amplified 164 bp product.
Confirmation of Male-specificity by PCR amplification and Gel electrophoresis
PCR reactions were performed with either purified buffalo genomic DNA or lysates from embryos. Embryonic lysates were prepared as described elsewhere (Appa Rao et al., 1993). Amplifications were carried out in a total volume of 25 ul of reaction mixture containing embryonic lysate as a template DNA. The reaction mixture contained PCR buffer, 1.5 mM of MgCLZ, 200 uM of each dNTP (dATP, dCTP, dGTP & dTTP), 20 pM of each primer, 1.25 U of Taq DNA polymerase and overlaid with mineral oil. Each embryonic lysate was divided into three tubes (a,b,c) and subjected to 'a' primary PCR with BRY.I primer, 'b1 with nested PCR with BuRYN.I primer and 'c' with multiplex PCR, where nested primers were co-amplified with ZFX/ZFY specific primers. The amplification were carried out in a DNA Thermal Cycler for 35 cycles following a standard cycling program (Appa Rao et al., 1993).
Positive (male and female DNA isolated buffalo from lymphocytes) and negative control (no DNA) were included during each batch of amplification to ensure no cross-contamination had occurred. Amplification products (about 15 ul) were electrophoresed on 1.8% agarose gel for 2 h, stained with ethidium bromide and visualized under ultraviolet light. The embryo was judged to be a male, if a 301 bp product from BRY.I was visible in the reaction 'a', 164 bp product with BuRYN.I hi the reaction 'b' and both 164 and 445 bp fragment in the reaction 'c'. If only a 445 bp fragment was visible in 'c' and no product is seen in both 'a' & 'b' reaction, the embryo was considered as a female. Phi X 174 DNA digested with Hae EH enzyme was run simultaneously as marker to assure that the amplified products were of expected size.
By choosing primers from Y-chromosome specific sequences, we amplified a 301 bp fragment, a BRY.I homologue designated as 'BuRY.I' from male genomic DNA of water buffalo (Bubalus bubalis). The PCR product amplified from male buffalo was excised from gel, purified and radioactively labelled and used as a probe to hybridize male specific amplification in buffalo. Southern blot analysis of PCR amplified products with probe BuRY.I, revealed only male-specific bands, thereby confirming the identity of the amplified fragment. Male specific amplifications were also seen in other species of bovidae family such as cattle, sheep and goat apart from buffalo (Fig. 1). The band intensity however, in lane 3 differ from that of other lanes, suggesting that BuRY.I sequence amplified from buffalo genome is not completely homologous to BRY.I sequence.
Further, hybridization of BuRY.I PCR product to Bam Hi-digests of buffalo male and female DNAs also demonstrate the male specificity of the sequence, as there were no prominant signals appeared hi female DNA, besides a weak band . Approximately 301 bp fragment corresponding to BuRY.I was successfully cloned
and sequenced (GenBank Accession No. X93551). Sequence analysis revealed 88% homology with BRY.I, besides several polymorphisms (Fig.2). TTiirty six pohit mutations (28 substitutions, 4 deletions and additions each) were detected in the 301 bp sequence.
Sex determination was performed for a total of 80 IVM and FVF derived embryos from buffalo using nested and multiplex PCR. All parameters and reagent concentrations were systematically adjusted such that 100 pg of DNA, equivalent to 2-4 embryonic cells, would be sufficient to yield amplification products of the predicted size in less than 6 hours for sex-determination. As shown in table. 1, 80 embryos in total were examined, of which 41 and 39 were classified as male and
Table. 1: Sex determination of buffalo embryos by Primary and Nested PCR

(Table Removed)
female respectively. The sex of each embryo was determined both by primary PCR using BRY.I primer and nested PCR with BuRYN.I primes. The results obtained were in complete agreement with each other, indicating the method is accurate in its detection, sensitive enough for this species and reliable.

1. A Y-chromosome specific DNA sequence, as shown below, wherein the said
sequence is capable of detecting y-chromosome sequence in buffalo, cattle, sheep
and goat,
2. A Y-chromosome specific DNA sequence and a method, substantially as herein






482-del-2002-description (complete).pdf










Patent Number 211365
Indian Patent Application Number 482/DEL/2002
PG Journal Number 45/2007
Publication Date 09-Nov-2007
Grant Date 26-Oct-2007
Date of Filing 22-Apr-2002
Applicant Address ARUNA ASAF ALI MARG, NEW DELHI 110067, INDIA.
# Inventor's Name Inventor's Address
PCT International Classification Number A61K 31/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA