公开(公告)号：US09868946B2

公开(公告)日：2018-01-16

申请号：US14440694

申请日：2013-11-01

Applicant: GIRIHLET INC.

Inventor： Anitha Jayaprakash ,  Ravi Sachidanandam

IPC: C12N9/16 ,  C12N9/14 ,  C12N9/22 ,  C12N1/08 ,  C12N15/10

CPC classification number: C12N15/1006 ,  C12N9/16 ,  C12N15/1003 ,  C12Y301/11005

Abstract: A method for preparing circular double stranded mitochondrial DNA (mtDNA) substantially free of genomic DNA (gDNA) comprising the steps of: providing a cellular lysate free of protein and RNA contaminants, precipitating cellular debris and proteins out of said lysate and obtaining a solution comprising purified circular double stranded mitochondrial DNA (mtDNA) and genomic DNA (gDNA), incubating said solution with an amount of Hind Exonuclease V for a time and at a temperature effective to cleave non-circular DNA and obtain circular double stranded mtDNA, incubating said circular double stranded mtDNA with an amount of Ampure beads effective to bind said circular double stranded mtDNA, washing said beads with ethanol, and eluting said mtDNA from said beads, wherein said method is free of ultra-centrifugation.

公开(公告)号：US20150275200A1

公开(公告)日：2015-10-01

申请号：US14440694

申请日：2013-11-01

Applicant: GIRIHLET INC

Inventor： Anitha Jayaprakash ,  Ravi Sachidanandam

IPC: C12N15/10 ,  C12N9/16

CPC classification number: C12N15/1006 ,  C12N9/16 ,  C12N15/1003 ,  C12Y301/11005

Abstract: A method for preparing circular double stranded mitochondrial DNA (mtDNA) substantially free of genomic DNA (gDNA) comprising the steps of: providing a cellular lysate free of protein and RNA contaminants, precipitating cellular debris and proteins out of said lysate and obtaining a solution comprising purified circular double stranded mitochondrial DNA (mtDNA) and genomic DNA (gDNA), incubating said solution with an amount of Hind Exonuclease V for a time and at a temperature effective to cleave non-circular DNA and obtain circular double stranded mtDNA, incubating said circular double stranded mtDNA with an amount of Ampure beads effective to bind said circular double stranded mtDNA, washing said beads with ethanol, and eluting said mtDNA from said beads, wherein said method is free of ultra-centrifugation.

Abstract translation: 一种制备基本上不含基因组DNA（gDNA）的环状双链线粒体DNA（mtDNA）的方法，包括以下步骤：提供不含蛋白质和RNA污染物的细胞裂解物，将细胞碎片和蛋白质从所述裂解物中沉淀出来并获得包含 纯化的圆形双链线粒体DNA（mtDNA）和基因组DNA（gDNA），将所述溶液与一定量的Hind外切核酸酶V孵育一段时间，并在有效切割非环状DNA的温度下获得圆形双链mtDNA，将所述环状 具有有效结合所述圆形双链mtDNA的Ampure珠的双链mtDNA，用乙醇洗涤所述珠粒，并从所述珠粒中洗脱所述mtDNA，其中所述方法不含超离心。