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Method for amplification and assay of RNA fusion gene variants, method of distinguishing same and related primers, probes, and kits

A fusion and gene technology, applied in the field of oligonucleotide primers and probes, kits

Inactive Publication Date: 2016-01-06
ABBOTT MOLECULAR INC A CORP ORGANISED & EXISTING UNDER THE LAWS OF THE STATE OF DELAWARE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, PML-RARα form S may be associated with worse prognosis compared with PML-RARα forms V and L

Method used

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  • Method for amplification and assay of RNA fusion gene variants, method of distinguishing same and related primers, probes, and kits
  • Method for amplification and assay of RNA fusion gene variants, method of distinguishing same and related primers, probes, and kits
  • Method for amplification and assay of RNA fusion gene variants, method of distinguishing same and related primers, probes, and kits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0193] This example describes the design of primers for BCR-ABL amplification.

[0194] Amplification involved the use of forward and reverse primers positioned on opposite sides of the translocation breakpoint between the fused BCR and ABL gene segments. Thus, each resulting BCR-ABL amplicon contained a variant-specific linker sequence. One reverse primer (RP) and three forward primers (FP) were prepared. The reverse primer a2RP anneals to a specific sequence within exon a2 of ABL that is shared by variants e1a2, b2a2, b3a2 and e19a2. The reverse primer a2RP directs reverse transcription of RNA from multiple variants. Forward primers e1FP, b2FP and e19FP anneal to specific sequences within exons e1, b2 and e19 of the BCR, respectively. The primer pair e1FP and a2RP will amplify the cDNA target sequence reverse transcribed from the variant e1a2 RNA. The primer pair b2FP and a2RP will amplify the cDNA target sequence reverse transcribed from the variant b2a2 and b3a2 RNA. ...

Embodiment 2

[0204] This example describes the design of the BCR-ABL probe.

[0205]Four short oligonucleotide probes were designed with high affinity to junction-specific sequence specificity from each of the targeted BCR-ABL variants (i.e., e1a2, b2a2, b3a2, and e19a2) to hybridize. The BCR-ABL exon junctions spanned by each probe are separated by intron sequences in the genomic DNA. Thus, the probe hybridizes only to amplicons derived from BCR-ABL RNA / cDNA. The specificity of the probe will enable discrimination of the variants. The probes can be used individually to detect a single BCR-ABL variant. Alternatively, two or more probes can be used together to detect multiple BCR-ABL variants in a single reaction. Probes can be labeled identically for combined detection of targeted BCR-ABL variants. Alternatively, for differential detection of two or more BCR-ABL variants in a single reaction, the probes can be labeled differently. The sequences of the probes are shown in Table IV.

...

Embodiment 3

[0224] This example describes the design of internal control (IC) amplification primers and probes.

[0225] Two oligonucleotide primers (ie, forward primer and reverse primer) were designed for amplification of each of the three endogenous genes used as internal controls. The primers were designed to amplify the well-characterized housekeeping genes β-glucuronidase (GUSB), ABL and glucose-6-phosphate dehydrogenase (G6PD).

[0226] The GUSB forward primer (GUSBFP) anneals to a sequence located in exon 8 of GUSB. The GUSB reverse primer (GUSBRP) anneals to a sequence located in exon 10 of GUSB, and in combination with GUSBFP directs the amplification of the reverse transcribed GUSB cDNA. The resulting amplicon spans the sequence from exons 8, 9 and 10 of GUSB.

[0227] Oligonucleotide probes were designed for hybridization and detection of GUSB amplicons. The probe was designed to hybridize to sequences in exon 9 of GUSB.

[0228] The ABL forward primer (ABLFP) anneals to a...

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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.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application No. 61 / 800,593, filed March 15, 2013, which is hereby incorporated by reference in its entirety. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been filed electronically in ASCII format, and is hereby incorporated by reference in its entirety. The ASCII copy (created on March 7, 2014) is named 11438WOO1_SEQ.txt and is 400,373 bytes in size. technical field [0005] The present disclosure relates to fusion gene variants, methods of amplifying polymerase chain reaction (PCR) such as real-time PCR, methods of distinguishing fusion gene variants, oligonucleotide primers and probes, and kits. Background technique [0006] Certain types of human leukemia are often associated with the presence of fusion genes (specifically, a fusion of the breakpoint cluster region (BCR) gene and the Abelson murine leukemia (AB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/16
Inventor S.X.黄J.D.维特施克
Owner ABBOTT MOLECULAR INC A CORP ORGANISED & EXISTING UNDER THE LAWS OF THE STATE OF DELAWARE
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