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Lna-based mutant enrichment next-generation sequencing assays

A DNA molecule and sequence technology, applied in the field of LNA-based mutation enrichment next-generation sequencing assay, which can solve the problem of complex tumor-specific mutations and other problems

Inactive Publication Date: 2018-02-16
THE GENERAL HOSPITAL CORP
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  • Abstract
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Problems solved by technology

However, detection of tumor-specific mutations from all these sources is complicated by the fact that mutations are exceptionally rare against the background of normal cellular DNA

Method used

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  • Lna-based mutant enrichment next-generation sequencing assays
  • Lna-based mutant enrichment next-generation sequencing assays
  • Lna-based mutant enrichment next-generation sequencing assays

Examples

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example

[0057] The invention is further illustrated in the following examples, which in no way limit the scope of the invention described in the claims.

[0058] method

[0059] The following method is used in the example shown below.

[0060] Genomic DNA (gDNA) from circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) was extracted using the Qiagen AllPrep DNA / RNA Micro Kit or Qiagen Circulating Nucleic Acid Kit, respectively, according to the manufacturer's protocol. Semifunctional sequencing libraries were prepared by combining DNA templates with new semifunctional gene-specific primers, matching gene-specific LNA clamps with the KAPA HiFi Hot-Start PCR Kit (KAPA Biosystems), and performing 25 rounds of primer extension, primer extension represents a critical step in mutant enrichment.

[0061] Next, 0.4X Solid Phase Reversible Immobilization (SPRI) bead cleanup was performed with Agencourt Ampure XP beads (Beckman Coulter, USA) according to the manufacturer's protocol...

example 1

[0078] Example 1. ESR1 and PIK3CA mutation analysis using enrichment sequencing

[0079] This example describes the development and exemplary implementation of the method described here as enrichment-sequencing, using locked nucleic acid clamps to recruit mutant-enriched set. A highly stringent, multiphase bioinformatics approach is then applied to ensure optimal specificity for mutation calling.

[0080] For the development of this technology, we first focused on estrogen receptor (ER)-positive breast cancers, in which recurrent mutations in the estrogen receptor alpha gene, ESR1, have recently been detected and appear to confer resistance to endocrine therapy (1-5). Identification of ESR1 mutations through noninvasive monitoring of women with metastatic breast cancer who have undergone endocrine therapy may allow early identification of treatment resistance, thereby allowing timely changes in therapy. Because mutations in ESR1 appear to cluster in the ligand-binding domai...

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Abstract

Ultra-sensitive assays for the detection of mutations, e.g., from blood-based sources of tumor genetic material (circulating tumor cells or plasma), or other settings in which limiting amounts of DNA,e.g., tumor DNA, is available. The assay is exemplified in the estrogen receptor, but is broadly customizable to target mutations in other genes.

Description

[0001] priority statement [0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 147,851, filed April 15, 2015, and U.S. Provisional Patent Application Serial No. 62 / 248,154, filed October 29, 2015. The entire contents of the above documents are hereby incorporated by reference. [0003] Federally Funded Research or Advancement [0004] This invention was made with government support under Grant No. CA129933 awarded by the National Institutes of Health. The US Government has certain rights in this invention. technical field [0005] An ultrasensitive PCR-based assay is described for the detection of mutations, e.g., from blood-based sources of tumor genetic material (circulating tumor cells or plasma), or in other settings where limited amounts of DNA (e.g., tumor DNA) are available. Determination. Background technique [0006] Analysis of tumor-derived genetic material from non-tissue-based sources is poised to revolutionize th...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/10C12Q1/6827
CPCC12Q1/6827A61P15/00A61P35/00C12Q2525/161C12Q2525/191C12Q2531/113C12Q2535/122C12Q2549/126C12N15/1058
Inventor T·K·桑德里森郑宗利D·A·哈伯S·马赫斯瓦兰A·J·亚弗拉特
Owner THE GENERAL HOSPITAL CORP
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