Methods of Identifying Homologous Genes Using FISH
First Claim
1. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprisingidentifying a first nucleotide type that is a single nucleotide polymorphism within the first gene,hybridizing a first primer type directly upstream of the first nucleotide type, extending the first primer type across the first nucleotide type in the presence of a first polymerase, first extension nucleotides and a first labeled extension nucleotide complementary to the first nucleotide type, wherein the first labeled extension nucleotide hybridizes to the first nucleotide type,identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type,hybridizing a second primer type directly upstream of the second nucleotide type, extending the second primer type across the second nucleotide type in the presence of a second polymerase, second extension nucleotides and a second labeled extension nucleotide complementary to the second nucleotide type wherein the second labeled extension nucleotide hybridizes to the second nucleotide type, wherein the first gene is differentially labeled from the second gene.
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Accused Products
Abstract
The present invention relates to methods of hybridizing nucleic acid probes to genomic DNA.
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Citations
52 Claims
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1. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that is a single nucleotide polymorphism within the first gene, hybridizing a first primer type directly upstream of the first nucleotide type, extending the first primer type across the first nucleotide type in the presence of a first polymerase, first extension nucleotides and a first labeled extension nucleotide complementary to the first nucleotide type, wherein the first labeled extension nucleotide hybridizes to the first nucleotide type, identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, hybridizing a second primer type directly upstream of the second nucleotide type, extending the second primer type across the second nucleotide type in the presence of a second polymerase, second extension nucleotides and a second labeled extension nucleotide complementary to the second nucleotide type wherein the second labeled extension nucleotide hybridizes to the second nucleotide type, wherein the first gene is differentially labeled from the second gene.
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18. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by sequences present in one gene and not the other and wherein the first gene and the second gene are homologs comprising
identifying sequence differences between the first gene and the second gene, hybridizing first labeled probes to sequences present in the first gene but not present in the second gene, hybridizing second labeled probes to sequences present in the second gene but not in the first gene, wherein the first label is different from the second label and the first gene is differentially labeled compared to the second gene.
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19. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that is a single nucleotide polymorphism within a first sequence within the first gene, hybridizing a first labeled complementary sequence to the first sequence and with a first labeled nucleotide of the first labeled complementary sequence hybridizing to the first nucleotide type, hybridizing a first complementary sequence to the first sequence and adjacent to the first labeled complementary sequence, ligating the first labeled complementary sequence to the first sequence, identifying a second nucleotide type that is a single nucleotide polymorphism within a second sequence within the second gene, hybridizing a second labeled complementary sequence to the second sequence and with a second labeled nucleotide of the second labeled complementary sequence hybridizing to the second nucleotide type, hybridizing a second complementary sequence to the second sequence and adjacent to the second labeled complementary sequence, ligating the second labeled complementary sequence to the second sequence, wherein the first gene is differentially labeled from the second gene.
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23. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that is a single nucleotide polymorphism within a first sequence within the first gene, hybridizing a first labeled complementary sequence to the first sequence and with a first labeled nucleotide of the first labeled complementary sequence hybridizing to the first nucleotide type, hybridizing a first complementary sequence to the first sequence and adjacent to the first labeled complementary sequence, ligating the first labeled complementary sequence to the first sequence, identifying a second nucleotide type that is a single nucleotide polymorphism within a second sequence within the second gene, hybridizing a second labeled complementary sequence to the second sequence and with a second labeled nucleotide of the second labeled complementary sequence hybridizing to the second nucleotide type, hybridizing a second complementary sequence to the second sequence and adjacent to the second labeled complementary sequence, ligating the second labeled complementary sequence to the second sequence, identifying a third nucleotide type that is a single nucleotide polymorphism within a third sequence within the first gene and which is different from the first nucleotide type and the second nucleotide type, hybridizing a third labeled complementary sequence to the third sequence and with a third labeled nucleotide of the third labeled complementary sequence hybridizing to the third nucleotide type, hybridizing a third complementary sequence to the third sequence and adjacent to the third labeled complementary sequence, ligating the third labeled complementary sequence to the third sequence, identifying a fourth nucleotide type that is a single nucleotide polymorphism within a fourth sequence within the first gene and which is different from the first nucleotide type, the second nucleotide type and the third nucleotide type, hybridizing a fourth labeled complementary sequence to the fourth sequence and with a fourth labeled nucleotide of the fourth labeled complementary sequence hybridizing to the fourth nucleotide type, hybridizing a fourth complementary sequence to the fourth sequence and adjacent to the fourth labeled complementary sequence, ligating the fourth labeled complementary sequence to the fourth sequence, and wherein the first gene is differentially labeled from the second gene.
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30. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that is a single nucleotide polymorphism within the first gene, hybridizing a first probe including a first labeled nucleotide complementary to the first nucleotide type, identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, hybridizing a second probe including a second labeled nucleotide complementary to the second nucleotide type, wherein the first gene is differentially labeled from the second gene.
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32. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that is a single nucleotide polymorphism within the first gene, hybridizing a first probe including a first labeled nucleotide complementary to the first nucleotide type, identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, hybridizing a second probe including a second labeled nucleotide complementary to the second nucleotide type, identifying a third nucleotide type that is a single nucleotide polymorphism within the first gene and which is different from the first nucleotide type and the second nucleotide type, hybridizing a third probe including a third labeled nucleotide complementary to the third nucleotide type, identifying a fourth nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, the second nucleotide type, and the third nucleotide type, hybridizing a fourth probe including a fourth labeled nucleotide complementary to the fourth nucleotide type, wherein the first gene is differentially labeled from the second gene.
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34. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that is a single nucleotide polymorphism within the first gene, hybridizing a first amplifiable probe to a first sequence including the first nucleotide type and including a first labeled nucleotide complementary to the first nucleotide type, amplifying the first amplifiable probe to produce one or more amplicons including the first labeled nucleotide, identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, hybridizing a second amplifiable probe to a second sequence including the second nucleotide type and including a second labeled nucleotide complementary to the second nucleotide type, amplifying the second amplifiable probe to produce one or more amplicons including the second labeled nucleotide, wherein the amplicons including the first nucleotide type are differentially labeled from the amplicons including the second nucleotide type.
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37. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that is a single nucleotide polymorphism within the first gene, hybridizing a first end and a second end of first amplifiable probe to first sequences flanking the first nucleotide type, extending and ligating nucleotides from the first end of the first amplifiable probe to the second end of the first amplifiable probe and across the first nucleotide type and including a first labeled nucleotide complementary to the first nucleotide type, amplifying the first amplifiable probe to produce one or more amplicons including the first labeled nucleotide, identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, hybridizing a first end and a second end of second amplifiable probe to second sequences flanking the second nucleotide type, extending and ligating nucleotides from the first end of the second amplifiable probe to the second end of the second amplifiable probe and across the second nucleotide type and including a second labeled nucleotide complementary to the first nucleotide type, amplifying the second amplifiable probe to produce one or more amplicons including the second labeled nucleotide, wherein the amplicons including the first nucleotide type are differentially labeled from the amplicons including the second nucleotide type.
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40. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that is a single nucleotide polymorphism within the first gene, hybridizing a first end and a second end of a first probe to first sequences flanking the first nucleotide type, extending and ligating nucleotides from the first end of the first probe to the second end of the first probe and across the first nucleotide type and including a first non-naturally occurring nucleotide complementary to the first nucleotide type, identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type, hybridizing a first end and a second end of a second probe to second sequences flanking the second nucleotide type, extending and ligating nucleotides from the first end of the second probe to the second end of the second probe and across the second nucleotide type and including a second non-naturally occurring nucleotide complementary to the first nucleotide type, wherein the first non-naturally occurring nucleotide is different from the second non-naturally occurring nucleotide.
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41. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by sequence differences which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that indicates a sequence difference within the first gene, hybridizing a first primer type directly upstream of the first nucleotide type, extending the first primer type across the first nucleotide type in the presence of a first polymerase, first extension nucleotides and a first labeled extension nucleotide complementary to the first nucleotide type, wherein the first labeled extension nucleotide hybridizes to the first nucleotide type, identifying a second nucleotide type that indicates a sequence difference within the second gene and which is different from the first nucleotide type, hybridizing a second primer type directly upstream of the second nucleotide type, extending the second primer type across the second nucleotide type in the presence of a second polymerase, second extension nucleotides and a second labeled extension nucleotide complementary to the second nucleotide type wherein the second labeled extension nucleotide hybridizes to the second nucleotide type, wherein the first gene is differentially labeled from the second gene.
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45. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by sequence differences which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first nucleotide type that indicates a sequence difference within the first gene, hybridizing a first primer type directly upstream of the first nucleotide type, extending the first primer type across the first nucleotide type in the presence of a first polymerase, first extension nucleotides and a first labeled extension nucleotide complementary to a nucleotide in the sequence difference, wherein the first labeled extension nucleotide hybridizes to the nucleotide in the sequence difference, identifying a second nucleotide type that indicates a sequence difference within the second gene and which is different from the first nucleotide type, hybridizing a second primer type directly upstream of the second nucleotide type, extending the second primer type across the second nucleotide type in the presence of a second polymerase, second extension nucleotides and a second labeled extension nucleotide complementary to a nucleotide in the sequence difference wherein the second labeled extension nucleotide hybridizes to the nucleotide in the sequence difference, wherein the first gene is differentially labeled from the second gene.
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49. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by sequence differences which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprising
identifying a first sequence difference within the first gene, hybridizing a first primer type directly upstream of the first sequence difference, extending the first primer type across the first sequence difference in the presence of a first polymerase, first extension nucleotides and a first labeled extension nucleotide complementary to a nucleotide in the first sequence difference, wherein the first labeled extension nucleotide hybridizes to the nucleotide in the first sequence difference, identifying a second sequence difference within the second gene and which is different from the first sequence difference, hybridizing a second primer type directly upstream of the second sequence difference, extending the second primer type across the second sequence difference in the presence of a second polymerase, second extension nucleotides and a second labeled extension nucleotide complementary to a nucleotide in the second sequence difference wherein the second labeled extension nucleotide hybridizes to the nucleotide in the second sequence difference, wherein the first gene is differentially labeled from the second gene.
Specification