Method for amplification and assay of rna fusion gene variants, method of distinguishing same and related primers, probes, and kits
AssigneeAbbott Molecular Inc.
InventorsShihai X. Huang, Jeffrey D. Wuitschick
Life of this patent
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Abstract
Method for amplification, alone or in further combination with detection or detection and quantitation, of RNA from fusion gene variants, method of distinguishing same, and oligonucleotide primers and probes and kits for use in the methods.
Claims36
A method of amplifying mRNA from variants of a fusion between a first gene and a second gene in a sample of mRNA, which method comprises:
- (a) (i) obtaining cDNA, which has been reverse-transcribed from the sample of mRNA, or reverse-transcribing the sample of mRNA to cDNA and (ii) amplifying the reverse-transcribed cDNA using primers comprising a primer for an exon of the first gene, which becomes contiguous with a variant exon of the second gene, and a primer for each of two or more variant exons of the second gene,
- wherein one or more primers can be detectably labeled,
- whereupon mRNA from variants of a fusion between a first gene and a second gene in a sample of mRNA is amplified.
A set of primers comprising at least two primers selected from the group consisting of a primer that hybridizes to exon e1 of BCR, a primer that hybridizes to exon b2 of BCR, and a primer that hybridizes to exon e19 of BCR, wherein the primer can be detectably labeled and/or wherein the set of primers is combined with at least one detectably labeled probe selected from the group consisting of a probe that hybridizes to a nucleotide sequence at or near a junction of a BCR-ABL gene fusion comprising the 3′ end of exon e1 of BCR and the 5′ end of exon a2 of ABL, a probe that hybridizes to a nucleotide sequence at or near a junction of a BCR-ABL gene fusion comprising the 3′ end of exon b2 of BCR and the 5′ end of exon a2 of ABL, a probe that hybridizes to a nucleotide sequence at or near a junction of a BCR-ABL gene fusion comprising the 3′ end of exon b3 of BCR and the 5′ end of exon a2 of ABL, and a probe that hybridizes to a nucleotide sequence at or near a junction of a BCR-ABL gene fusion comprising the 3′ end of exon e19 of BCR and the 5′ end of exon a2 of ABL.
A set of probes comprising at least two probes selected from the group consisting of a probe that hybridizes to a nucleotide sequence at or near a junction of a BCR-ABL gene fusion comprising the 3′ end of exon e1 of BCR and the 5′ end of exon a2 of ABL, a probe that hybridizes to a nucleotide sequence at or near a junction of a BCR-ABL gene fusion comprising the 3′ end of exon b2 of BCR and the 5′ end of exon a2 of ABL, a probe that hybridizes to a nucleotide sequence at or near a junction of a BCR-ABL gene fusion comprising the 3′ end of exon b3 of BCR and the 5′ end of exon a2 of ABL, and a probe that hybridizes to a nucleotide sequence at or near a junction of a BCR-ABL gene fusion comprising the 3′ end of exon e19 of BCR and the 5′ end of exon a2 of ABL.
A kit comprising:
- (i) a set of primers comprising a primer that hybridizes to an exon of a first gene, which becomes contiguous with a variant exon of a second gene, and a primer for each of two or more variant exons of the second gene;
- and
- (ii) instructions for a method of detecting mRNA from fusions of the first gene and the second gene in a sample of mRNA, which method comprises:
- (a) (i′) obtaining cDNA, which has been reverse-transcribed from the sample of mRNA, or reverse-transcribing the sample of mRNA to cDNA and (ii′) amplifying the reverse-transcribed cDNA using primers comprising a primer for an exon of the first gene, which becomes contiguous with a variant exon of the second gene, and a primer for each of two or more variant exons of the second gene, wherein one or more of the primers can be detectably labeled, and
- (b) detecting the amplified cDNA or detecting and quantitating the amplified cDNA.
Dependencies are inferred from the claim text.