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Compositions and methods for detection of nucleic acid mutations

A technology of nucleotides and universal primers, applied in biochemical equipment and methods, combinatorial chemistry, nucleotide libraries, etc., can solve problems such as inability to apply plasma fusion detection

Inactive Publication Date: 2019-03-01
NATERA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gene fusions are commonly detected by mRNA-Seq in tumor biopsies, but this method cannot be applied to fusion detection in plasma

Method used

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  • Compositions and methods for detection of nucleic acid mutations
  • Compositions and methods for detection of nucleic acid mutations
  • Compositions and methods for detection of nucleic acid mutations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0165] Example 1. Identification of fusion gene breakpoints for tiling analysis

[0166] Provided herein are examples of how to design and select a series of tiling primers for use in the methods of the invention, particularly methods for detecting gene fusions using single-sided nested PCR reactions. Design of tiling primers for detection of gene fusions begins with mapping COSMIC fusion transcripts to genomic coordinates (ie, translocations). However, it was found that the use of transcript-level information caused uncertainty in breakpoint locations, as most were rearrangements of introns (and thus spliced ​​out of transcripts). Therefore, it was necessary to cover a range of sequences for each reported fusion based on exon boundaries. Identification of molecular markers may aid in the development of cancer panels for the identification of gene fusions and may be applied to other cancers and diseases beyond lung cancer, eg ALK in hematopoietic, lymphoid tissue, RET in thyr...

Embodiment 2A

[0178] Example 2A. Development of synthetic fusion standards

[0179] Design of Gene Fusion Probes

[0180] As used herein, fusion probes refer to synthetic gene fusions such as CD74:Ros1, NMP1:Alk1(x2) and TPM4:Alk1. The first genes are partners (such as CD74, NMP1 and TPM4) and the latter are targets (Ros1, ALK1 (both groups) and Alk1). Fusion probes were designed to correspond to the average length of cfDNA, a length of 160 bp was chosen. Such as figure 1 As shown in , the design utilizes nine tiling primers spanning the 160 fusion probe "target".

[0181] As used herein, fusion probes are designed to span a "junction," which can refer to a fusion break between two fusion partners. To illustrate, consider the following example, there are two genes A and B consisting of the sequences {a_i} and {b_i} between which a fusion occurs. To generate fusion probes, we first identified the location of the breakpoint in each gene and then constructed probe S:

[0182] S=a_{i-m},....

Embodiment 2B

[0184] Example 2B. Development of synthetic fusion standards

[0185] The design of synthetic fusion probes is done to develop a system that allows detection of gene fusion profiles. Identification of gene fusion profiles can aid in the identification of fusion genomic sequences for rearrangement following sequencing of the fusion genomic DNA. Genomic sequences (suspected to have gene fusions) were used to construct tiling primer template synthetic oligonucleotides tiling across each target sequence containing breakpoints as tiling fusion probes, each 160 bp in length . figure 1 The tiling of these synthetic oligonucleotides for construction of fusion probes is illustrated.

[0186] A literature review of published translocation genome sequences to identify gene fusion products. This led to the selection of six regions (5 ALK, 1 ROS) containing gene fusions, followed by bioinformatics calculations to identify the corresponding genomic positions to unify the results.

[018...

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Abstract

The invention provides methods and compositions for detecting a mutation in a target gene in a sample of blood or a fraction thereof, including in certain examples, a fraction that includes circulating tumor DNA. The methods can include a tiling PCR reaction, for example a one-sided multiplex tiling reaction. Virtually any type of mutation can be detected with the methods and compositions. In certain embodiments, gene fusions are detected. Improved PCR methods, especially for performing nested multiplex PCR reactions are provided.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 357,847, filed July 01, 2016, which is incorporated herein by reference in its entirety. [0003] sequence listing [0004] This application contains a Sequence Listing that has been submitted electronically in ASCII format, and is hereby incorporated by reference in its entirety. The ASCII copy was created on June 29, 2017, was named N_017_WO_01_SL.TXT, and was 83,468 bytes in size. technical field [0005] The disclosed invention generally relates to methods for detecting nucleic acid mutations and fusions using amplification methods such as polymerase chain reaction (PCR). Background technique [0006] Detection of mutations associated with disease, including cancer, whether prior to diagnosis, in making a diagnosis for disease staging, or to monitor treatment response, has traditionally relied on solid tumor biopsy samples. Such sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6827C12Q2531/113C12Q2535/122C12Q2537/143C12Q1/6853C12Q1/686C12Q1/6886C12Q2600/112C12Q2600/118C12Q2600/156C12Q2600/16C40B40/06G01N2800/7028
Inventor 伯恩哈德·齐默尔曼约书亚·巴比亚尔兹拉赫勒·萨拉里图德·庞皮利厄·康斯坦丁奥努尔·萨卡里亚丹尼斯·普罗森亚历山大·奥尔森斯科特·达什纳尼古拉·谢尔盖耶夫马修·迈卡·希尔
Owner NATERA
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