Invention Grant
- Patent Title: Integrated purification and measurement of DNA methylation and co-measurement of mutations and/or MRNA expression levels in an automated reaction cartridge
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Application No.: US15182394Application Date: 2016-06-14
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Publication No.: US11603555B2Publication Date: 2023-03-14
- Inventor: Edwin W. Lai , Andrew Kohlway , Reuel Vanatta , Russell Higuchi , Alexander Gall , Kriszten Kocmond
- Applicant: Cepheid
- Applicant Address: US CA Sunnyvale
- Assignee: Cepheid
- Current Assignee: Cepheid
- Current Assignee Address: US CA Sunnyvale
- Agency: Weaver Austin Villeneuve & Sampson LLP
- Agent Emily M. Haliday
- Main IPC: C12Q1/6806
- IPC: C12Q1/6806 ; C12Q1/6827 ; C12Q1/6886 ; C12N15/10 ; B01L7/00

Abstract:
In various embodiments methods of determining methylation of DNA are provided. In one illustrative, but non-limiting embodiment the method comprises i) contacting a biological sample comprising a nucleic acid to a first matrix material comprising a first column or filter where said matrix material binds and/or filters nucleic acids in said sample and thereby purifies the DNA; ii) eluting the bound DNA from the first matrix material and denaturing the DNA to produce eluted denatured DNA; iii) heating the eluted DNA in the presence of bisulfite ions to produce a deaminated nucleic acid; iv) contacting said deaminated nucleic acid to a second matrix material comprising a second column to bind said deaminated nucleic acid to said second matrix material; v) desulfonating the bound deaminated nucleic acid and/or simultaneously eluting and desulfonating the nucleic acid by contacting the deaminated nucleic acid with an alkaline solution to produce a bisulfite converted nucleic acid; vi) eluting said bisulfite converted nucleic acid from said second matrix material; and vii) performing methylation specific PCR and/or nucleic acid sequencing, and/or high resolution melting analysis (HRM) on said bisulfite-converted nucleic acid to determine the methylation of said nucleic acid, wherein at least steps iv) through vi) are performed in a single reaction cartridge.
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