Detection of Fusion Events Using Replicate PCR Reactions

a technology of fusion events and pcr, applied in the field of gene fusion events, can solve the problems of only being able to detect gene fusion, fish cannot be applied to cells or tissues, and the approach cannot be applied to mrna

Inactive Publication Date: 2019-08-08
INIVATA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present patent is about methods for detecting genomic rearrangements, particularly gene fusion events, by targeting a DNA molecule of interest with a set or pool of primers. The primers produce an amplification product when a genomic rearrangement is present, which can then be sequenced to identify the presence and position of the rearrangement. The methods do not require a further enrichment step and can detect multiple genomic rearrangements in a single reaction. The methods are also advantageous because they do not require end repair or ligation to enrich for targets of interest and there is no loss of starting material due to processing prior to fusion detection.

Problems solved by technology

The technical problem addressed in this patent is how to efficiently detect gene fusions in cancer, especially those occurring at a low allelic frequency, while minimizing costs and simplifying the process. Current methods involve various limitations including difficulty in identifying all relevant breaks, being restricted to certain types of data, and having poor accuracy in determining the location of fusions. There is a need for more effective tools to improve early detection and classification of disease states based on genomic analysis.

Method used

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  • Detection of Fusion Events Using Replicate PCR Reactions
  • Detection of Fusion Events Using Replicate PCR Reactions
  • Detection of Fusion Events Using Replicate PCR Reactions

Examples

Experimental program
Comparison scheme
Effect test

example 1

of EML4-ALK Variant at a Range of Allelic Fractions

[0499]A custom cell free DNA reference standard containing an EML4-ALK fusion of sequence GAAGTTCCTATACTTTCTAGAGAATAGGAACTTC (SEQ ID NO: 1) at an allelic fraction of 2.5% was obtained from Horizon Discoveries. This reference standard was diluted in sheared (average 188 bp) human placental DNA (Bioline) to achieve allelic fractions of 1%, 0.5%, 0.25%, 0.125% and 0.0625%. Three samples were created at each allelic fraction.

[0500]Each sample was split into two replicates, each containing a total of 4000 input copies. PCR amplification was performed on two replicates using the ALK primer panel (table 1). Each PCR contained 25 uL DNA, 27.5 uL Platinum SuperFi 2× Master Mix (Invitrogen) and 2.5 uL of the ALK primer pool (for primer concentration see table 1). PCR cycling was followed using manufacturer' instructions. The PCR product was cleaned up using SPRIselect reagent (Beckman Coulter B23319) using the manufacturers protocol. DNA was ...

example 2

of ROS1-CD74 Variant

[0502]A synthetic gBlock containing a ROS1 fusion sequence (based on a sequence reported in the literature: Seki, Mizukami and Kohno, Biomolecules, 2015, 5, 2464-2476) was synthesized by IDT and was sheared using the covaris to achieve an average size of 150 bp. The gBlock was added to sheared (average 188 bp) human placental DNA (Bioline) to achieve an allelic fraction of 1%.

[0503]Each sample was split into two replicates, each containing a total of 4000 input copies. PCR amplification was performed on two of the replicates using the ROS1 primer panel (table 2). Each PCR contained 25 uL DNA, 27.5 uL Platinum SuperFi 2× Master Mix (Invitrogen) and 2.5 uL of the ROS1 primer pool (for primer concentration see table 2). PCR Cycling was followed using manufactures instructions. The PCR product was cleaned up using SPRIselect reagent (Beckman Coulter B23319) using the manufacturers protocol. DNA was eluted in 18 uL and a second PCR using Indexed illumina primers was p...

example 3

of ROS1-CD74 Variant Using Sequential Amplification

[0505]The same synthetic ROS1 fusion gBlock at 1% allelic fraction as was used in Example 2 was tested. Each sample was split into two replicates, each containing a total of 4000 input copies. Linear amplification of the template was performed on two of the replicates using only the ROS1 forward primer panel. Each reaction contained 25 uL DNA, 27.5 uL Platinum SuperFi 2× Master Mix (Invitrogen) and 2.5 uL of the ROS1 forward primer pool. Cycling was followed using manufactures instructions. The PCR product was cleaned up once using SPRIselect reagent (Beckman Coulter B23319) using the manufacturers protocol. DNA was eluted in 18 uL and a first PCR using a i5 adapter forward primer and the ROS1 reverse primer pool was performed. Each PCR contained 10 uL DNA, 25 uL Platinum SuperFi 2× Master Mix (Invitrogen), 2.5 ul of the i5 adapter forward primer and 2.5 uL of the ROS1 reverse primer pool. Cycling was followed using manufactures ins...

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Abstract

The present disclosure relates to methods for detecting and targeting genomic rearrangements, in particular gene fusion events, by targeting a DNA molecule of interest with a set or pool of primers, wherein the forward primers and reverse primers produce a PCR amplification product when a genomic rearrangement is present. The present disclosure also relates to methods of bioinformatic analysis to determine whether or not the detection of an amplification product from the selective PCR is actually indicative of the presence of a gene fusion. The present disclosure also related to related methods of diagnosis and treatment of diseases and conditions associated with such genomic rearrangements, in particular cancers, such as lung cancer.

Description

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Claims

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Application Information

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Owner INIVATA LTD
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