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 7,435,794 B2
• Filed: 05/12/2004
• Issued: 10/14/2008
• Est. Priority Date: 12/04/2001
• Status: Expired due to Term

• Current Assignee: Evrogen Jsc
• Original Assignee: Evrogen Jsc
• Inventors: Lukyanov, Sergey A., Shagin, Dmitriy A., Rebrikov, Denis V.
• Primary Examiner(s): Strzelecka; Teresa E.
• Application Number:
  US10/845,366
• Publication Number:
  US 20050164216A1
• Time in Patent Office: 1,616 Days
• Field of Search: None
• US Class Current: 530/350
• 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. An isolated divalent cation dependent thermostable nuclease with 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.

2. The nuclease according to claim 1 wherein said nuclease hydrolyzes single stranded nucleic acids and RNA in duplex nucleic acids with lower activity than DNA in duplex nucleic acids.

3. The nuclease according to claim 1 wherein said nuclease cleaves deoxyribonucleic acid molecules in short completely matched duplex nucleic acids with at least 10-fold higher activity than in non-completely matched duplex nucleic acids of the same length.

4. The nuclease according to claim 1 wherein said nuclease is isolated from the Kamchatka crab.

5. The nuclease according to claim 2 wherein said nuclease hydrolyzes single stranded nucleic acids and RNA in duplex nucleic acids with at least 10-fold lower activity than DNA in duplex nucleic acids.

6. The nuclease according to claim 1 wherein said nuclease is recombinant.

7. A method of selectively cleaving deoxyribonucleic acid molecules in duplex nucleic acids in a complex nucleic acid sample, said method comprising:

contacting said sample with a nuclease according to claim 1 under DSN conditions for a period of time sufficient for said duplex nucleic acids in said sample to be selectively cleaved.

8. The method according to claim 7, wherein said method comprises distinguishing duplex deoxyribonucleic acids from single stranded deoxyribonucleic acids.

9. The method according to claim 7, wherein said method comprises distinguishing completely matching duplex deoxyribonucleic acids that include at least one deoxyribonucleic acid molecule from non-completely matching duplex deoxyribonucleic acids of the same length.

10. A kit for use in performing a method according to claim 7, wherein said kit comprises:

a nuclease according to claim 1; and

instructions for practicing a method according to claim 7.