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Methods for assessing risk using total and specific cell-free DNA

A cell-free, specific technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve limited and other problems

Pending Publication Date: 2019-08-09
威斯康星州立大学医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current methods for quantitative analysis of nucleic acid populations are limited

Method used

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  • Methods for assessing risk using total and specific cell-free DNA
  • Methods for assessing risk using total and specific cell-free DNA
  • Methods for assessing risk using total and specific cell-free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 - Mismatch Amplification Assay (MOMA)

[0127] SNV target selection

[0128] As presently described, identification of targets for multiplexing according to the present disclosure may comprise one or more of the following steps. First, highly heterozygous SNPs can be screened on several ethnic control populations (Hardy-Weinberg p > 0.25), excluding known regions of difficulty. Difficult regions include syndromic regions that may be abnormal in patients and regions of low complexity, including centromeres and telomeres of chromosomes. Target fragments of the desired length can then be designed in silico. Specifically, two 20 to 26 bp primers spanning a 70 bp window for each SNP can be designed. All candidate primers can then be queried to GCRh37 using BLAST. Those primers found to be sufficiently specific can be retained and monitored for off-target hits, especially at the 3' end of the fragment. Off-target candidate hits can be analyzed for generation of...

Embodiment 3

[0146] Example 3 - Determination of total cell-free DNA

[0147] In some embodiments, total cell-free DNA (cf-DNA) is determined. Three to ten milliliters (ml) of anticoagulated blood were collected in 10 ml cell-free DNA blood collection tube (BCT) tubes (Streck, Omaha, NE). Plasma was separated from whole blood by centrifugation and stored at -80°C until DNA extraction. ReliaPrep available TM HT Circulating Nucleic Acid Kit, Custom (Promega, Madison, WI) was used for cf-DNA extraction.

[0148] Total cf-DNA content from plasma was assessed in triplicate by quantitative real-time PCR as previously described (Hidestrand et al., Influence of temperature during transportation on cell-free DNA analysis. Fetal Diagn Ther 31, 122-128 (2012)). PCR analysis was performed on an Applied Biosystems QuantStudio7 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA).

Embodiment 4

[0149] Example 4 - Risk assessment using total cell-free DNA and specific cell-free DNA

[0150] Such as Figure 7 As shown, values ​​for total cell-free nucleic acid (eg, DNA) and values ​​for specific cell-free nucleic acid (eg, DNA) can be displayed together at multiple time points in the report.

[0151] Figure 9 Another example of a report showing two types of data is shown. Data from 68 patients were analyzed. As expected, when both total cell-free DNA and specific cell-free DNA values ​​were considered high, it indicated that rejection was likely to occur and drive an inflammatory process (upper right quadrant) or poor outcome or prognosis, such as death. When total cell-free DNA is considered high but specific cell-free DNA is not high, infection rather than rejection is likely to occur in the subject (upper left quadrant). However, when total cell-free DNA is considered low while specific cell-free DNA is considered high, it is likely that the subject is in the e...

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Abstract

This invention relates to methods and compositions for assessing risk by measuring total and specific cell-free nucleic acids (such as DNA) in a subject. The methods and compositions provided herein can be used to determine risk of a condition, such as transplant rejection.

Description

[0001] Cross References to Related Applications [0002] This application is based on 35 U.S.C. §119(e), §120 and §365(c) requirements of U.S. Provisional Application No. 62 / 416,689 filed on November 2, 2016 and International Application No. filed on April 29, 2017 .PCT / US2017 / 030293, the respective contents of which are hereby incorporated by reference in their entirety. technical field [0003] The present invention relates to methods and compositions for assessing risk by measuring total and specific cell-free nucleic acid (eg, cell-free DNA) in a subject. The methods and compositions provided herein can be used to determine the risk of conditions such as graft rejection or other adverse graft outcomes. Background technique [0004] The ability to detect and quantify low levels of nucleic acid in a sample may allow early detection of conditions such as graft rejection or other adverse transplant outcomes. However, current methods for quantitative analysis of nucleic aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6809
CPCC12Q1/6809C12Q1/6851C12Q1/6883C12Q2600/156C12Q2537/165C12Q2545/114C12Q2525/185C12Q2535/125C12Q2537/143C12Q2560/00C12Q2600/00
Inventor 青·富田·米切尔迈克尔·米切尔卡尔·施塔姆
Owner 威斯康星州立大学医学院
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