公开(公告)号：US11260387B2

公开(公告)日：2022-03-01

申请号：US15839731

申请日：2017-12-12

Applicant: Cepheid

Inventor： Edwin Wei-Lung Lai ,  Andrew Kohlway ,  Reuel Van Atta ,  Russell Higuchi ,  Alexander A. Gall ,  Kriszten Kocmond

IPC: B01L3/00 ,  B01L7/00 ,  C12Q1/6827 ,  C12N15/10 ,  C12Q1/6853 ,  C12Q1/686

Abstract: 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) desulphonating the bound deaminated nucleic acid and/or simultaneously eluting and desulphonating 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.

公开(公告)号：US20220226809A1

公开(公告)日：2022-07-21

申请号：US17586645

申请日：2022-01-27

Applicant: Cepheid

Inventor： Edwin Wei-Lung Lai ,  Andrew Kohlway ,  Reuel Van Atta ,  Russell Higuchi ,  Alexander A. Gall ,  Kriszten Kocmond

IPC: B01L3/00 ,  B01L7/00 ,  C12Q1/6827 ,  C12N15/10 ,  C12Q1/6853 ,  C12Q1/686

Abstract: 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) desulphonating the bound deaminated nucleic acid and/or simultaneously eluting and desulphonating 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.

公开(公告)号：US11603555B2

公开(公告)日：2023-03-14

申请号：US15182394

申请日：2016-06-14

Applicant: Cepheid

Inventor： Edwin W. Lai ,  Andrew Kohlway ,  Reuel Vanatta ,  Russell Higuchi ,  Alexander Gall ,  Kriszten Kocmond

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.

公开(公告)号：US20240141415A1

公开(公告)日：2024-05-02

申请号：US18095982

申请日：2023-01-11

Applicant: Cepheid

Inventor： Edwin Wei-Lung Lai ,  Andrew Kohlway ,  Reuel Van Atta ,  Russell Higuchi ,  Alexander A. Gall ,  Kriszten Kocmond

IPC: C12Q1/6827 ,  B01L7/00 ,  C12N15/10 ,  C12Q1/6806 ,  C12Q1/6886

CPC classification number: C12Q1/6827 ,  B01L7/52 ,  C12N15/1006 ,  C12N15/1017 ,  C12Q1/6806 ,  C12Q1/6886 ,  B01L2300/0864 ,  B01L2300/087 ,  C12Q2600/154

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.