Procedure for the sex determination of embryos in mammals especially applied to bovine embryos
First Claim
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1. A process for sex determination of embryos in bovines which comprises:
- a) isolating a bovine embryo at the blastula stage;
b) cutting a zone of cells away from the pellucidae membrane of said embryo by microsurgery and extracting a sample of about 8 to about 19 cells from said zone of cells;
c) separating said about 8 to about 19 cells from remaining traces of the embryo'"'"'s pellucidae membrane;
d) isolating DNA from said about 8 to about 19 cells by placing the sample in a tube with water, heating the contents of the tube to 95°
C. to form an aqueous denatured DNA solution, immediately chilling the contents of said tube by placing the tube in ice, and centrifuging the contents of said tube to recover any condensed liquid containing the aqueous denatured DNA solution;
e) performing a first round of a polymerage chain reaction (PCR) on said aqueous denatured DNA solution to amplify a first fragment of DNA wherein said first fragment of DNA is a subsequence of a sequence in a homologous genome region of the X and Y chromosome, using a first pair of oligonucleotide primers with the sequence 5'"'"'-ATAATCACATGGAGAGCCACAAGCT-3'"'"' (SEQ. ID. NO;
3) and 5'"'"'-CGACTTCTTTGGTATCTGAGAAAGT-3'"'"' (SEQ. ID. NO;
4), in the presence of a DNA polymerage enzyme and nucleotides until a solution of a first amplified DNA fragment is obtained;
f) obtaining an aliquot of said solution and subjecting said aliquot to a second round of PCR to amplify a second DNA fragment whose sequence is a subsequence of said first amplified DNA fragment using a second pair of oligonucleotide primers with the sequence;
5'"'"'-TTGAATGTGATGAGTGTGGG-3'"'"' (SEQ. ID. NO;
5) and 5'"'"'-AAGTCAGAAGACAAATGTCA-3'"'"' (SEQ. ID. NO;
6) until a solution of a second amplified DNA fragment is obtained;
g) digesting said second amplified DNA fragment with PstI, wherein the presence of a PstI cut site in said second amplified DNA fragment indicates the presence of a Y chromosome and wherein the absence of a PstI cut site in said second amplified DNA fragment indicates the presence of an X chromosome, thereby forming a mixture of cut and/or uncut second amplified DNA fragments;
h) size separating said mixture of cut and/or uncut second amplified DNA fragments by electrophoresis on a 2% agarose gel and determining the size of digested fragments by a comparison of bands obtained on said gel with a standard molecular weight marker wherein the presence of a 378 base pair band is indicative of the presence of an X chromosome and the presence of two bands which are 293 base pairs and 85 base pairs each is indicative of the presence of a Y chromosome;
i) determining the sex of the embryo based on results obtained in step (h).
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Abstract
A procedure is provided for the sex determination of embryos in mammals, that is particularly exemplified on bovines. The procedure overcomes the limitations given in former techniques. It includes a new stage of polymerase chain reaction PCR method, using specific oligonucleotides in order to amplify fragments corresponding to the sampled cells and not from false ones, spuriously produced. In this way, the necessary sensitivity is obtained. The oligonucleotides sequences are particularly selected for the procedure. The procedure is used for the sex determination of embryos in mammals, and the procedure is sensitive enough to be carried out a sample containing few cells, is highly secure, and which give positive results for both female and male sex; specially applied to bovine embryos.
46 Citations
2 Claims
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1. A process for sex determination of embryos in bovines which comprises:
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a) isolating a bovine embryo at the blastula stage; b) cutting a zone of cells away from the pellucidae membrane of said embryo by microsurgery and extracting a sample of about 8 to about 19 cells from said zone of cells; c) separating said about 8 to about 19 cells from remaining traces of the embryo'"'"'s pellucidae membrane; d) isolating DNA from said about 8 to about 19 cells by placing the sample in a tube with water, heating the contents of the tube to 95°
C. to form an aqueous denatured DNA solution, immediately chilling the contents of said tube by placing the tube in ice, and centrifuging the contents of said tube to recover any condensed liquid containing the aqueous denatured DNA solution;e) performing a first round of a polymerage chain reaction (PCR) on said aqueous denatured DNA solution to amplify a first fragment of DNA wherein said first fragment of DNA is a subsequence of a sequence in a homologous genome region of the X and Y chromosome, using a first pair of oligonucleotide primers with the sequence 5'"'"'-ATAATCACATGGAGAGCCACAAGCT-3'"'"' (SEQ. ID. NO;
3) and 5'"'"'-CGACTTCTTTGGTATCTGAGAAAGT-3'"'"' (SEQ. ID. NO;
4), in the presence of a DNA polymerage enzyme and nucleotides until a solution of a first amplified DNA fragment is obtained;f) obtaining an aliquot of said solution and subjecting said aliquot to a second round of PCR to amplify a second DNA fragment whose sequence is a subsequence of said first amplified DNA fragment using a second pair of oligonucleotide primers with the sequence;
5'"'"'-TTGAATGTGATGAGTGTGGG-3'"'"' (SEQ. ID. NO;
5) and 5'"'"'-AAGTCAGAAGACAAATGTCA-3'"'"' (SEQ. ID. NO;
6) until a solution of a second amplified DNA fragment is obtained;g) digesting said second amplified DNA fragment with PstI, wherein the presence of a PstI cut site in said second amplified DNA fragment indicates the presence of a Y chromosome and wherein the absence of a PstI cut site in said second amplified DNA fragment indicates the presence of an X chromosome, thereby forming a mixture of cut and/or uncut second amplified DNA fragments; h) size separating said mixture of cut and/or uncut second amplified DNA fragments by electrophoresis on a 2% agarose gel and determining the size of digested fragments by a comparison of bands obtained on said gel with a standard molecular weight marker wherein the presence of a 378 base pair band is indicative of the presence of an X chromosome and the presence of two bands which are 293 base pairs and 85 base pairs each is indicative of the presence of a Y chromosome; i) determining the sex of the embryo based on results obtained in step (h). - View Dependent Claims (2)
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Current AssigneeHilding Ohlsson SA
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Original AssigneeHilding Ohlsson SA
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InventorsUgalde, Rodolfo A., Fr asch, Alberto C.
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Primary Examiner(s)Zitomer, Stephanie W.
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Assistant Examiner(s)REES, DIANE
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Application NumberUS08/425,711Time in Patent Office586 DaysField of Search436/6, 435/91.2, 435/78US Class Current435/6.11CPC Class CodesC12Q 1/6879 for sex determinationC12Q 2600/156 Polymorphic or mutational m...
