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US 7,459,271 B2Granted
Method for detecting whether an organism is homozygous or heterozygous using labelled primers and RFLP
AssigneeMurdoch Childrens Research Institute
InventorKirby Siemering
Life of this patent
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Abstract
The invention is a method for detecting whether an organism is homozygous or heterozygous in a target nucleotide. The method combines hybridization of two differently labelled probes A and B on either side of a restriction site, restriction endonuclease digestion, and detection of the labels in the resulting digest. If an organism is: homozygous then the labels will be present in the ratio 1:1 or 1:0; or if heterozygous then the labels will be present in the ratio 1:2. Either immobilized array technology or electrophoretic separation may be used.
Claims10
A method for determining the presence or absence of a homozygous or heterozygous mutation in a gene, said method comprising:
- amplifying a target nucleotide sequence within the gene using a first and a second primer to produce an amplified product, wherein said first primer comprises one or more chemically modified nucleotides, bases or phosphodiester bonds such that a nucleotide strand which extends from said first primer is substantially resistant to exonuclease activity, and wherein said second primer comprises a nucleotide sequence which introduces in the strand extended by said first primer a tag sequence which is complementary to a capture oligonucleotide immobilized to a solid support and wherein one or both of said first or second primers introduces a restriction site in the target sequence when there is no mutation;
- digesting the amplified product with an exonuclease to digest the strand not amplified by the primer comprising the exonuclease-resistant nucleotides, bases or phosphodiester linkages to generate a single-stranded nucleic acid molecule;
- hybridizing to said single-stranded nucleic acid molecule a probe that contains complementarity to a region encompassing the restriction site that is introduced when no mutation is present to generate a partial double-stranded molecule wherein the probe comprises two reporter molecules capable of facilitating the provision of identifiable signals which can be distinguished from each other;
- contacting the partially double-stranded molecules with a restriction endonuclease which will digest the double-stranded molecules which do not contain the mutation and subjecting digested and/or non-digested molecules to conditions to permit annealing of the tag sequence to the capture oligonucleotide immobilized to the solid support;
- measuring the relative intensity of the signal by the reporter molecules;
- wherein an equal intensity of different signals represents a homozygous presence of the mutation;
- wherein one signal represents an absence of the mutation;
- and
- wherein a mixture of signals, in which the intensity of one signal is less than the intensity of the other signal thereby represents a heterozygous presence of the mutation.
6 more elements
A method for determining the presence or absence of a homozygous or heterozygous mutation in a gene, said method comprising:
- amplifying a target nucleotide sequence within the gene using a first and second primer to produce an amplified product, wherein said first primer comprises one or more chemically modified nucleotides, bases or phosphodiester bonds such that a nucleotide strand which extends from said first primer is substantially resistant to exonuclease activity, and wherein said second primer comprises a nucleotide sequence which introduces in the strand extended by said first primer a tag sequence which is complementary to a capture oligonucleotide immobilized to a solid support and wherein one or both of said first or second primers removes a restriction site in the target sequence when there is no mutation;
- digesting the amplified product with an exonuclease to digest the strand not amplified by the primer comprising the exonuclease-resistant nucleotides, bases or phosphodiester linkages to generate a single-stranded nucleic acid molecule;
- hybridizing to said single-stranded nucleic acid molecule a probe that contains complementarity to a region encompassing the restriction site that is removed when no mutation is present to generate a partial double-stranded molecule wherein the probe comprises two reporter molecules capable of facilitating the provision of identifiable signals which can be distinguished from each other;
- contacting the partially double-stranded molecules with a restriction endonuclease which will digest the double-stranded molecules which contain the mutation and subjecting digested and/or non-digested molecules to conditions to permit annealing of the tag sequence to the capture oligonucleotide immobilized to the solid support;
- measuring the relative intensity of the signal by the reporter molecules wherein an equal intensity of different signals represents a homozygous absence of the mutation;
- wherein one signal represents a homozygous mutation;
- and
- wherein a mixture of signals, in which the intensity of one signal is less than the intensity of the other signal thereby represents a heterozygous presence of the mutation.
5 more elements
Dependencies are inferred from the claim text.