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Fusion gene microarray

A technology that combines genes and microarrays, applied in the determination/testing of microorganisms, biochemical equipment and methods, etc.

Inactive Publication Date: 2010-06-16
UNIV OSLO HF
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In other words, sequence-specific RT-PCR is required for analytical function, and the above reasons in turn limit the throughput of the method

Method used

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  • Fusion gene microarray
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Experimental program
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Effect test

Embodiment 1

[0112] Preparation of ligation probes (oligonucleotides) and microarrays

[0113] To generate ligation probes (oligonucleotides (oligos)), we produced computer scripts (written in the Python programming language) that automatically process public genomic data. We used the www.biomart.org Internet portal to obtain exon sequences for all genes and their transcripts. For each combination of fusion genes, the end sequence (last 30 nucleotides) of all gene A exons and the start sequence (30 nt) of all gene B exons were combined in all combinations. Next, an oligonucleotide calculation algorithm was used to generate probes (oligonucleotides) from these potential fusion gene exon-to-exon junctions. Here we optimize the Tm at 68°C and keep the Tm equal on each side of the junction. In our example, we generated exon-to-exon junction probes (oligonucleotides) that were 33-46 nucleotides in length.

[0114] In this way 47427 ligation probes (oligonucleotides) were designed for 275 fus...

Embodiment 2

[0119] functioning microarray

[0120] In a proof-of-principle experiment, we analyzed a set of positive control samples where each fusion gene was known to be present. Test samples included four prostate cancer tissue samples positive for the TMPRSS2:ERG fusion gene and two leukemia cell lines, each known to carry one of the TCF3:PBX1 fusion gene and the ETV6:RUNX1 fusion gene.

[0121] For test samples, total RNA was isolated by using a Qiagen spin column. In addition, ribosomal RNA reduction kit (RiboMinus TM Transcript Isolation Kit; Invitrogen) enriches for mRNA. From this, first-strand cDNA synthesis was performed with random primers (hexamers), followed by double-stranded cDNA and shipped to Nimblegen Inc. for labeling, hybridization, washing, and scanning of microarrays. cDNA was labeled by inclusion of Cy3- and Cy5-modified dNTPs.

[0122] in conclusion

[0123] To visualize the measurement of the positive control gene, we performed two separate pathways using ch...

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Abstract

The present invention relates to a microarray comprising a chimeric probe for an intergenic exon-to-exon junction of a fusion gene and at least two intragenic probes for a fusion gene partner of the fusion gene. The invention further relates to a method of detecting a fusion gene and a kit suitable for detecting fusion genes.

Description

[0001] background [0002] Cancer genomes often contain fusion genes that arise following rearrangements of chromosomal structure, such as translocations, deletions, and inversions. Fusion genes are commonly found in blood cancers. Fusion genes have now been found to be only infrequently associated with solid tumors, in contrast to the detection of substantial genomic copy number imbalances. However, recent reports have shown that fusion transcripts can often be shown to also contribute to solid tumorigenesis (Mitelman et al., 2007 (Mitelman et al., 2007), Teixeira, 2006 (Teixeira, 2006), Tomlins et al., 2005 (Tomlins et al., 2005)). A major problem has been the technical limitation of detecting fusion genes in solid tumors. [0003] Recently, certain fusion genes have been identified for differential diagnosis or treatment decisions in blood cancers and some rare solid tumor types. Currently, laboratory routine diagnostics employ cumbersome but inefficient assays to detect ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6837C12Q1/6886C12Q2600/156C12Q2600/158C12Q2539/105
Inventor 朗希尔德·A.·洛特古罗·E.·林德罗尔夫·I.·舍特海姆托马森·加德.O.S.罗格纳斯·托尔比约恩
Owner UNIV OSLO HF
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