Genotyping by mass spectrometric analysis of allelic fragments
First Claim
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1. A method for genotyping a diploid organism, comprising:
- providing two alleles of a target gene of a diploid organism suspected to contain a polymorphism;
obtaining a first segment of one allele wherein the segment contains the suspected polymorphic locus;
obtaining a second segment from the other allele wherein the segment also contains the suspected polymorphic locus;
replacing a natural nucleotide at greater than 90% of its points of occurrence in the first and the second segment with a modified nucleotide to give a first and a second modified segment;
wherein, the natural nucleotide that is replaced is not a nucleotide involved in the polymorphism;
replacing the natural nucleotide with a modified nucleotide comprises amplification using a primer that hybridizes to each segment such that, after amplification, a first modified nucleotide is incorporated between the 3′
end of the primer and the polymorphic locus; and
, a second modified nucleotide is located from 5 to 20 nucleotides downstream of the first modified nucleotide;
cleaving the first and second modified segments at greater than 90% of the points of occurrence of the modified nucleotide to give a first and second set of fragments each of which comprises a 5-20 nucleotide fragment; and
, comparing masses of the 5-20 nucleotide fragments obtained from the first and the second modified segment to detect the presence or absence of the polymorphism.
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Abstract
The present invention relates to a method for genotyping a diploid organism by cleaving segments of two alleles such that 7-20 nucleotide fragments that contain a suspected polymorphic locus are produced and comparing the masses of those fragments.
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Citations
21 Claims
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1. A method for genotyping a diploid organism, comprising:
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providing two alleles of a target gene of a diploid organism suspected to contain a polymorphism;
obtaining a first segment of one allele wherein the segment contains the suspected polymorphic locus;
obtaining a second segment from the other allele wherein the segment also contains the suspected polymorphic locus;
replacing a natural nucleotide at greater than 90% of its points of occurrence in the first and the second segment with a modified nucleotide to give a first and a second modified segment;
wherein,the natural nucleotide that is replaced is not a nucleotide involved in the polymorphism;
replacing the natural nucleotide with a modified nucleotide comprises amplification using a primer that hybridizes to each segment such that, after amplification, a first modified nucleotide is incorporated between the 3′
end of the primer and the polymorphic locus; and
,a second modified nucleotide is located from 5 to 20 nucleotides downstream of the first modified nucleotide;
cleaving the first and second modified segments at greater than 90% of the points of occurrence of the modified nucleotide to give a first and second set of fragments each of which comprises a 5-20 nucleotide fragment; and
,comparing masses of the 5-20 nucleotide fragments obtained from the first and the second modified segment to detect the presence or absence of the polymorphism. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 17, 18, 19, 20, 21)
the masses of the 7-20 nucleotide fragments obtained from the first and the second modified segment are compared to detect the presence or absence of the polymorphism.
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3. The method of claim 1, wherein the second modified nucleotide is from 7 to 12 nucleotides downstream of the first modified nucleotide;
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the masses of the 7-12 nucleotide fragments obtained from the first and the second modified segment are compared to detect the presence or absence of the polymorphism.
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4. The method of claim 1, wherein, if there would be less than 5 nucleotides between the first and second modified nucleotides, the method further comprises using a primer that contains a point mutation that removes the site of incorporation of either one of the modified nucleotides.
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5. The method of claim 1, wherein, if there are would be less than 7 nucleotides between the first and the second modified nucleotides, the method further comprises using a primer that contains a point mutation that removes the site of incorporation of either one of the modified nucleotides.
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6. The method of claim 1, wherein, if there would be more than 20 nucleotides between the first and second modified nucleotides, the method further comprises a primer, which contains a point mutation that incorporates a modified nucleotide downstream of the first modified nucleotide or upstream of the second modified nucleotide.
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7. The method of claim 1, wherein, if there would be more than 12 nucleotides between the first and the second modified nucleotides, the method further comprises a primer, which contains a point mutation that incorporates a modified nucleotide downstream of the first modified nucleotide or upstream of the second modified nucleotide.
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8. The method of claim 1, further comprising replacing a nucleotide known to occur at the polymorphic site with a mass-modified nucleotide.
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9. The method of claim 1, wherein comparing the masses of the fragments comprises using a mass spectrometer.
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10. The method of claim 9, wherein the mass spectrometer is a MALDI mass spectrometer.
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11. The method of claim 10, wherein the MALDI mass spectrometer is a MALDI-TOF mass spectrometer.
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12. The method of claim 9, wherein the mass spectrometer is an ESI mass spectrometer.
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16. The method of either claim 1 or claim 13, wherein comparing the masses of the fragments comprises using a mass spectrometer.
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17. The method of claim 16, wherein the mass spectrometer is a MALDI mass spectrometer.
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18. The method of claim 17, wherein the MALDI mass spectrometer is a MALDI-TOF mass spectrometer.
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19. The method of claim 16, wherein the mass spectrometer is an ESI mass spectrometer.
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20. The method of either claim 1 or claim 13, wherein the percentage replacement of a natural nucleotide with a modified nucleotide, the percentage cleavage at a modified nucleotide, or both the percentage replacement and the percentage cleavage, is greater than 95%.
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21. The method of either claim 1 or claim 13, wherein the percentage replacement of a natural nucleotide with a modified nucleotide, the percentage cleavage at a modified nucleotide, or both the percentage replacement and the percentage cleavage, is greater than 99%.
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13. A method for genotyping a diploid organism, comprising:
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providing two alleles of a target gene of a diploid organism suspected to contain a polymorphism;
obtaining a first segment of one allele wherein the segment contains the suspected polymorphic locus;
obtaining a second segment from the other allele wherein the segment also contains the suspected polymorphic locus;
replacing a natural nucleotide at greater than 90% of its points of occurrence in the first and the second segment with a modified nucleotide to give a first and a second modified segment;
wherein,the natural nucleotide that is replaced is a nucleotide involved in the polymorphism;
replacing the natural nucleotide with a modified nucleotide comprises amplification using a primer that hybridizes to each segment such that, after amplification, the suspected polymorphic locus is the first site of incorporation of a modified nucleotide after the end of the primer; and
,a second modified nucleotide is located from 5 to 20 nucleotides downstream of the first modified nucleotide;
cleaving the first and second modified segments at greater than 90% of the points of occurrence of the modified nucleotide to give a first and second set of fragments; and
,comparing masses of the two sets of fragments for the presence of the 5-20 nucleotide fragment wherein, if the fragment is present or absent in both sets, the gene is homozygous and if the fragment is present in only one set, the gene is heterozygous. - View Dependent Claims (14, 15)
the masses of the two sets of fragments obtained from the first and the second modified segment are compared to detect the presence of the 7-20 nucleotide fragment.
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15. The method of claim 13, wherein the second modified nucleotide is from 7 to 12 nucleotides downstream of the first modified nucleotide;
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the masses of the two sets of fragments obtained from the first and the second modified segment are compared to detect the presence of the 7-12 nucleotide fragment.
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Specification