Methods for quantitative genetic analysis of cell free DNA

a cell-free dna and quantitative technology, applied in the field of cell-free dna quantitative genetic analysis, can solve the problems of lack of reliable and robust molecular analysis methods in the analysis of genetic diseases, dna sequencing as a molecular diagnostic tool has been generally limited to the coding exons of one or two genes, and requires direct access to tumor tissues

Inactive Publication Date: 2016-02-25
RESOLUTION BIOSCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The art lacks reliable and robust molecular analysis methods for the analysis of genetic diseases.
Until recently, DNA sequencing as a molecular diagnostic tool has been generally limited to the coding exons of one or two genes.
While DNA sequencing has been used in the diagnosis and treatment of solid cancers, one of the most significant drawbacks of these methods is that they require direct access to tumor tissues.
Such material is often difficult to obtain from the initial biopsy used to diagnose the disease and virtually impossible to obtain in multiple repetitions over time.
Similarly, biopsies are not possible in patients with inaccessible tumors and not practical in individuals suffering from metastatic disease.
To date, existing molecular diagnostics approaches lack efficient solutions to clone and amplify individual DNA molecules, as well as solutions to efficiently target sequencing to specific genomic loci, with sensitivity sufficient to discriminate true positive test results from false positive signals that arise during sample processing.

Method used

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  • Methods for quantitative genetic analysis of cell free DNA
  • Methods for quantitative genetic analysis of cell free DNA
  • Methods for quantitative genetic analysis of cell free DNA

Examples

Experimental program
Comparison scheme
Effect test

example 1

Accurate Detection of Rare Mutations using Targeted Sequence Capture Technology

Purpose

[0221]The purpose of this experiment was to provide a direct proof-of-principle demonstration of rare variant detection using targeted sequence capture technology.

Background

[0222]Target sequence capture technology provides quantitative, sequence-based genetic analysis of nucleic acids and can be exploited to perform a combined mutational and copy number analysis of drug metabolism genes. The present inventors used targeted sequence capture technology and subsequence genetic analysis to detect rare sequence variants.

[0223]Genomic DNA inputs play a central role in rare variant detection, but quantitative analysis and control of genomic inputs places bounds on the estimated sensitivity of rare variant analysis. A genomic qPCR assay was used by the present inventors to estimate genomic inputs.

[0224]One experimental goal for rare variant analysis is to introduce 10-fold more genomic input as the target ...

example 2

A Novel Probe Design Effective for Comprehensive Sequencing of Target Regions in Highly Fragmented gDNA

Purpose

[0234]The purpose of these experiments is to develop an assay system to reliably and reproducibly interrogate circulating DNAs.

Background

[0235]Analysis of circulating DNA from body fluids represents an exciting, but as yet, unrealized opportunity in molecular diagnostics. Genomic DNA is highly intact. Literature suggest that the average size of circulating DNA is about 150 bp, which correlates well to the size of DNAs wrapped around a single nucleosomal histone complex.

Summary

[0236]The technical parameters of targeted sequence capture technology contemplated herein were designed to accommodate highly fragmented DNA and to retain the ability to generate comprehensive sequence coverage of targeted DNA. Capture probe density was increased and the length of capture probe sequences was reduced from 60 nucleotide to 40 nucleotide to minimize uninformative sequence generation in th...

example 3

Genetic Analysis of Circulating DNA

Purpose

[0272]The purpose of this example was to benchmark the genetic analysis of cfDNA using an efficient cloning procedure for cfDNA and target retrieval system.

Background

[0273]While there is tremendous enthusiasm in the scientific and health-care community for “liquid biopsies”—analysis of circulating DNA (cfDNA) for markers associated with potential disease states, there is remarkably little practical information about this potential analyte.

Summary

[0274]Plasma samples collected from healthy donors and individuals suffering from either ovarian or colon cancers were used to perform the genetic analysis of circulating DNA. The amount and the overall character of circulating cfDNA can vary widely from individual to individual. Surprisingly, the present inventors found that cfDNA is readily clonable with an efficiency indistinguishable from highly purified and fragmented genomic DNA; that the fragment size was remarkably consistent, with an average...

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Abstract

The invention provides a method for genetic analysis in individuals that reveals both the genetic sequences and chromosomal copy number of targeted and specific genomic loci in a single assay. The present invention further provides methods for the sensitive and specific detection of target gene sequences and gene expression profiles.

Description

STATEMENT REGARDING SEQUENCE LISTING[0001]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is CLFK—002—00US_ST25.txt. The text file is 117 KB, was created on Aug. 22, 2014, and is being submitted electronically via EFS-Web.BACKGROUND[0002]1. Technical Field[0003]The invention relates generally to compositions and methods for the quantitative genetic analysis of cell free DNA (cfDNA). In particular, the present invention relates to improved targeted sequence capture compositions and methods for the genetic characterization and analysis of cfDNA.[0004]2. Description of the Related Art[0005]It is becoming increasing clear that most, if not all, of the most common human cancers are diseases of the human genome. The emerging picture is that somatic mutations accumulate during an individual's lifetime, some of wh...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G06F19/00G16B20/10G16B20/20G16B25/10G16B30/10
CPCC12Q1/6806G06F19/20G06F19/345G06F19/28G06F19/22C12Q1/6869G16B20/00G16B30/00G16B25/00G16B30/10G16B20/20G16B25/10G16B20/10C12Q2525/191C12Q2545/114C12Q2563/159
Inventor RAYMOND, CHRISTOPHER K.LIM, LEE P.ARMOUR, CHRISTOPHER D.
Owner RESOLUTION BIOSCI
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