Methods and kits for fragmenting DNA

Patent metadata

Field	Value
Publication number	US-11845928-B2
Application number	US-201615557502-A
Country	US
Kind code	B2
Filing date	Apr 14, 2016
Priority date	May 4, 2015
Publication date	Dec 19, 2023
Grant date	Dec 19, 2023

Abstract

A method of DNA fragmentation is provided. The method comprising incubating a semi-solid biological sample which comprises the DNA with an auxiliary domain-directed nuclease having a binding affinity and selectivity to pre-defined sites in the DNA so as to yield a DNA fragment-of-interest, to thereby fragment the DNA.

First claim

What is claimed is:

1. A method of fragmenting mammalian or plant DNA, the method comprising:
(a) providing a semi-solid biological sample which comprises lysed mammalian or plant cells in an agarose gel; and
(b) contacting said semi-solid biological sample with Cas9 clease and an RNA oligonucleotide- having a binding affinity and selectivity to pre-defined sites in the mammalian or plant DNA of said lysed mammalian or plant cells under conditions which allow sufficient enzyme diffusion in said agarose gel in the absence of pulsed-field gel electrophoresis and hybridization of the RNA oligonucleotide to the mammalian or plant DNA, so as to digest said mammalian or plant DNA and yield a DNA fragment-of-interest, wherein said fragment of interest is 50-200 kb in length.

2. The method of claim 1 , wherein said DNA is genomic DNA.

3. The method of claim 2 , wherein said DNA is chromosomal DNA.

4. The method of claim 1 , wherein said DNA is human DNA.

5. The method of claim 1 , wherein said semi-solid biological sample prevents DNA shearing.

6. The method of claim 1 , further comprising separating the DNA fragment-of-interest from the mammalian or plant DNA following step (b).

7. The method of claim 6 , wherein said separating the DNA fragment-of-interest comprises pulsed-field gel electrophoresis.

8. The method of claim 6 , wherein said separating is effected by at least one of:
(a) melting said semi-solid biological sample; and
(b) subjecting said semi-solid biological sample to enzymatic treatment which digests a gel matrix of said semi solid biological sample.

9. The method of claim 1 , wherein said semi-solid biological sample is in the form of a plug.

10. The method of claim 1 , wherein said contacting is effected for two hours.

11. A method of fragmenting mammalian or plant DNA, the method comprising:
(a) providing a semi-solid biological sample which comprises lysed mammalian or plant cells in an agarose gel of about 1%; and
(b) contacting said semi-solid biological sample with Cas9 nuclease and an RNA oligonucleotide having a binding affinity and selectivity to pre-defined sites in the mammalian or plant DNA of said lysed mammalian or plant cells under conditions which allow sufficient Cas9 nuclease diffusion in said agarose gel in the absence of pulsed-field gel electrophoresis, so as to digest said mammalian or plant DNA and yield a DNA fragment-of-interest.

12. The method of claim 11 , wherein said fragment of interest is 50-200 kb in length.

Assignees

• Univ Tsinghua

Inventors

• Zhu Ting
• Ebenstein Yuval
• Lou Chunbo
• Jiang Wenjun
• Zhao Xuejin
• Gabrieli Tslil

Classifications

• C12N15/1003 Primary

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12N15/101

  by chromatography, e.g. electrophoresis, ion-exchange, reverse phase · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2521/301

  Endonuclease · CPC title