Enzymes for the detection of nucleic acid sequences

• US 20030134349A1
• Filed: 01/11/2001
• Published: 07/17/2003
• Est. Priority Date: 05/24/2000
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Lyamicheva, Natalie E., Neri, Bruce P., Schaefer, James J., Lyamichev, Victor I., Kaiser, Michael W., Allawi, Hatim Taysir, Ma, Wu-Po
• Granted Patent: US 6,635,463 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to novel enzymes designed for direct detection, characterization and quantitation of nucleic acids, particularly RNA. The present invention provides enzymes that recognize specific nucleic acid cleavage structures formed on a target RNA sequence and that cleave the nucleic acid cleavage structure in a site-specific manner to produce non-target cleavage products. The present invention provides enzymes having an improved ability to specifically cleave a DNA member of a complex comprising DNA and RNA nucleic acid strands.

Claims

1. A composition comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

279 and the complement of SEQ ID NO;

279.

2. A vector comprising the nucleic acid sequence of claim 1.

3. A host cell comprising the vector of claim 2.

4. A composition comprising a polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NO:

279 and sequences that hybridize to the complement of SEQ ID NO;

279 under stringent conditions.

5. The composition of claim 4, wherein said polypeptide comprises SEQ ID NO:

280.

6. A method for producing an altered enzyme with improved functionality in a nucleic acid cleavage assay comprising:

a) providing;

i) a polypeptide comprising SEQ ID NO;

280;

ii) a nucleic acid encoding a polypeptide of SEQ ID NO;

280; and

iii) a nucleic acid test substrate;

b) introducing one or more heterologous domains into said nucleic acid to produce an altered nucleic acid encoding an altered enzyme;

c) contacting said altered enzyme and said polypeptide comprising SEQ ID NO;

280 with said nucleic acid test substrate to produce cleavage products; and

d) comparing cleavage product contacted with said altered enzyme to cleavage products contacted with said polypeptide comprising SEQ ID NO;

280.