Methods and compositions for selectively cleaving DNA containing duplex nucleic acids in a complex nucleic acid mixture, and nuclease compositions for use in practicing the same

• US 20080138825A1
• Filed: 01/30/2008
• Published: 06/12/2008
• Est. Priority Date: 12/04/2001
• Status: Active Grant

• Current Assignee: Evrogen Jsc
• Original Assignee: Evrogen Jsc
• Inventors: Shagin, Dmitriy, Lukyanov, Sergey, Rebrikov, Denis V.
• Granted Patent: US 7,803,922 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10S 435/975 Kit

Abstract

Methods of selectively cleaving DNA containing duplex nucleic acids in a complex nucleic acid mixture, as well as nuclease containing compositions for use therein, are provided. In the subject methods, a nuclease or composition thereof is employed to provide for selective cleavage of DNA containing duplex nucleic acids in a complex nucleic acid mixture. Also provided are novel duplex-stranded specific nucleases and nucleic acids encoding the same, where the subject nucleases are enzymes that, preferentially cleave deoxyribonucleic acid molecules in perfectly matched nucleic acid duplexes as compared to non-perfectly matched nucleic acid duplexes of the same length and/or single stranded nucleic acids. The subject methods and compositions for practicing the same find use in a variety of different applications, including, but not limited to, nucleic acid analyte detection applications, gene expression profiling applications, detection of nucleic acid variants including single nucleotide polymorphisms applications, preparation of subtracted and normalized nucleic acid libraries, etc. Finally, kits for use in practicing the subject methods are provided.

Claims

1-37. -37. (canceled)

38. An isolated nucleic acid encoding a nuclease having at least 95% amino acid sequence identity to the amino acid sequence of a nuclease encoded by the nucleic acid comprising SEQ ID NO:

02.

39. The isolated nucleic acid of claim 38, said nucleic acid comprising the nucleotide sequence of SEQ ID NO:

02.

40. An isolated nucleic acid encoding a nuclease, wherein said nucleic acid hybridizes under stringent conditions with a nucleic acid according to claim 38, wherein said stringent hybridization conditions comprise a temperature of 50°

C. and 0.1×

SSC.

41. An expression cassette comprising:

a transcriptional initiation region functional in an expression host;

a nucleic acid having a nucleotide sequence found in a nucleic acid according to claim 38 under the transcriptional regulation of said transcriptional initiation region; and

a transcriptional termination region functional in said expression host.

42. A cell comprising an expression cassette according to claim 41 as part of an extrachromosomal element or integrated its genome as a result of introduction of said expression cassette into said cell.

43. A method of producing a nuclease, said method comprising:

growing a cell according to claim 42 to express said nuclease; and

isolating said nuclease substantially free of other proteins.

44. An expression cassette comprising:

a transcriptional initiation region functional in an expression host,

a nucleic acid having a nucleotide sequence found in a nucleic acid according to claim 40 under the transcriptional regulation of said transcriptional initiation region; and

a transcriptional termination region functional in said expression host.

45. A cell comprising an expression cassette according to claim 44 as part of an extrachromosomal element or integrated its genome as a result of introduction of said expression cassette into said cell.

46. A method of producing a nuclease, said method comprising:

growing a cell according to claim 45 to express said nuclease; and

isolating said nuclease substantially free of other proteins.