Oligonucleotides for genotyping thymidylate synthase gene
AssigneeF. Hoffmann-La Roche AG
InventorsHideyuki Yasuno, Kazushige Mori
Life of this patent
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Abstract
Oligonucleotides for genotyping the thymidylate synthase gene are provided. The number of tandem repeats in the promoter region of the thymidylate synthase gene can be identified based on the hybridization of an oligonucleotide of the invention to the genomic DNA of a subject. Therefore, the genotype of the thymidylate synthase gene can be identified based on the number of tandem repeats. The genotype relates to the responsiveness of a subject towards an antitumor agent.
Claims62
A method for identifying a number of tandem repeats in the promoter region of a thymidylate synthase gene, the method comprising:
- (a) amplifying a genomic DNA that comprises tandem repeats in at least the promoter region of the thymidylate synthase gene;
- (b) contacting, under stringent hybridization conditions, the amplified genomic DNA with either (i) a first oligonucleotide comprising the nucleotide sequence of SEQ ID NO:1 and a second oligonucleotide comprising the nucleotide sequence of SEQ ID NO:2, or (ii) a first oligonucleotide comprising the nucleotide sequence of the exact complement of SEQ ID NO:1 and a second oligonucleotide comprising the nucleotide sequence of the exact complement of SEQ ID NO:2, wherein the hybridization conditions are such that hybridization between the first oligonucleotide and the amplified genomic DNA can be detected if three tandem repeats are present, but not if only two tandem repeats are present;
- (c) detecting whether hybridization between the first oligonucleotide and the amplified genomic DNA has occurred;
- and
- (d) identifying the number of tandem repeats as “two” when hybridization is not detected.
A method for identifying a number of tandem repeats in the promoter region of a thymidylate synthase gene, the method comprising:
- (a) amplifying a genomic DNA that comprises tandem repeats in at least the promoter region of the thymidylate synthase gene;
- (b) contacting, under stringent hybridization conditions, the amplified genomic DNA with either (i) a first oligonucleotide comprising the nucleotide sequence of SEQ ID NO:1 and a second oligonucleotide comprising the nucleotide sequence of SEQ ID NO:2, or (ii) a first oligonucleotide comprising the nucleotide sequence of the exact complement of SEQ ID NO:1 and a second oligonucleotide comprising the nucleotide sequence of the exact complement of SEQ ID NO:2, wherein the hybridization conditions are such that hybridization between the first oligonucleotide and the amplified genomic DNA can be detected if three tandem repeats are present, but not if only two tandem repeats are present;
- (c) detecting whether hybridization between the first oligonucleotide and the amplified genomic DNA has occurred;
- (d) contacting the first and second oligonucleotides with the amplified genomic DNA of step (a) under hybridization conditions that are less stringent than in step (b);
- (e) detecting whether hybridization between the first oligonucleotide and the amplified genomic DNA has occurred in (d);
- and
- (f) identifying the number of tandem repeats as “two” when hybridization is not detected in (c), but is detected in (e).
A method for identifying a number of tandem repeats in the promoter region of a thymidylate synthase gene, the method comprising:
- (a) amplifying a genomic DNA that comprises tandem repeats in at least the promoter region of the thymidylate synthase gene;
- (b) hybridizing a sample of the amplified genomic DNA with either (i) a first oligonucleotide comprising the nucleotide sequence of SEQ ID NO:1 and a second oligonucleotide comprising the nucleotide sequence of SEQ ID NO:2, or (ii) a first oligonucleotide comprising the nucleotide sequence of the exact complement of SEQ ID NO:1 and a second oligonucleotide comprising the nucleotide sequence of the exact complement of SEQ ID NO:2;
- (c) subjecting the sample to melting curve analysis to determine the melting temperature (Tm);
- (d) comparing the Tm determined in (c) to the Tm obtained with the first oligonucleotide in each of two controls:
- a two tandem repeat (2R) control and a three tandem repeat (3R) control;
- and
- (e) identifying the number of tandem repeats as “two” when the Tm determined in (c) is more similar to the Tm obtained in the two tandem repeat control than to the Tm obtained in the three tandem repeat control.
A method for identifying a number of tandem repeats in the promoter region of a thymidylate synthase gene, the method comprising:
- (a) amplifying a genomic DNA that comprises tandem repeats in at least the promoter region of the thymidylate synthase gene;
- (b) hybridizing a sample of the amplified genomic DNA with either (i) a first oligonucleotide comprising the nucleotide sequence of SEQ ID NO:1 and a second oligonucleotide comprising the nucleotide sequence of SEQ ID NO:2, or (ii) a first oligonucleotide comprising the nucleotide sequence of the exact complement of SEQ ID NO:1 and a second oligonucleotide comprising the nucleotide sequence of the exact complement of SEQ ID NO:2;
- (c) subjecting the sample to melting curve analysis to determine whether the sample's melting temperature (Tm) corresponds to a two tandem repeat Tm, a three tandem repeat Tm, or a combination thereof.
Dependencies are inferred from the claim text.