Thermostable ligase mediated DNA amplification system for the detection of genetic diseases
0 Assignments
0 Petitions
Accused Products
Abstract
The present invention relates to the cloning of the gene of a thermophilic DNA ligase, from Thermus aquaticus strain HB8, and the use of this ligase for the detection of specific sequences of nucleotides in a variety of nucleic acid samples, and more particularly in those samples containing a DNA sequence characterized by a difference in the nucleic acid sequence from a standard sequence including single nucleic acid base pair changes, deletions, insertions or translocations.
153 Citations
19 Claims
-
1. The cell line AK76 designated as ATCC 55032.
-
2. A plasmid selected from pDZ1 and designated as ATCC 68307, and pDZ7 and designated as ATCC 68308.
-
3. An expression vector comprising a DNA sequence encoding for a thermostable ligase selected from the group consisting of (1) Thermus aquaticus HB8 ligase;
- (2) a thermostable ligase having at least 6 sequential amino acid residues corresponding to 6 sequential amino acid residues in Thermus aquaticus HB8 ligase; and
(3) a ligase active mutant of Thermus aquaticus HB8 ligase wherein an amino acid residue has been inserted, substituted or deleted in or from the amino acid sequence of the ligase.
- (2) a thermostable ligase having at least 6 sequential amino acid residues corresponding to 6 sequential amino acid residues in Thermus aquaticus HB8 ligase; and
-
4. An isolated nucleic acid molecule encoding a thermostable ligase enzyme, wherein the nucleic acid molecule is selected from the group consisting of:
-
the nucleic acid encoding the amino acid of SEQ. ID. No. 2; the nucleic acid sequence set out in SEQ ID No. 1; and nucleic acid molecules which hybridize under stringent conditions of 2×
SSC, 40% formamide at 40°
C. to the nucleic acid sequence set out in SEQ ID No. 1. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11)
-
-
12. An isolated nucleic acid molecule comprising a nucleic acid sequence which hybridizes under stringent conditions of 2×
- SSC, 40% formamide at 40°
C. to the nucleic acid sequence set out in SEQ. ID. No. 1 and wherein said nucleic acid molecule encodes a ligase enzyme which has a biological property of catalyzing the formation of a phosphodiester bond at the site of a single-stranded break in duplex DNA. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
- SSC, 40% formamide at 40°
Specification