Thermostable ligase-mediated DNA amplifications system for the detection of genetic disease

• US 5,494,810 A
• Filed: 11/22/1994
• Issued: 02/27/1996
• Est. Priority Date: 05/03/1990
• Status: Expired due to Term

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Barany, Francis, Hood, Leroy, Zebala, John, Kaiser, Robert J. Jr., Nickerson, Deborah
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): GRIMES, ERIC BURTON
• Application Number:
  US08/343,785
• Time in Patent Office: 462 Days
• Field of Search: 435/6, 435/91.2, 435/91.52
• US Class Current: 435/91.52
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

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 in a ligase chain reaction (LCR) assay 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.

Claims

1. A method for amplifying a first nucleotide sequence and a second nucleotide sequence which are complementary and together form separate strands of a double stranded DNA molecule, said method comprising:

providing a sample containing the first nucleotide sequence and the second nucleotide sequence;

providing a first oligonucleotide set of at least two oligonucleotides suitable for ligation together at a first ligation junction and for hybridization without mismatch at the first ligation junction to the first nucleotide sequence, wherein the at least two oligonucleotides hybridize adjacent to one another on the first nucleotide sequence and have a hybridization temperature of about 50°

C. to 85°

C.;

providing a second oligonucleotide set of at least two oligonucleotides suitable for ligation together at a second ligation junction and for hybridization without mismatch at the second ligation junction to the second nucleotide sequence, wherein the at least two oligonucleotides of the second oligonucleotide set hybridize adjacent to one another on the second nucleotide sequence and have a hybridization temperature of about 50°

to 85°

C.;

providing a thermostable ligase which does not become irreversibly denatured and lose its catalytic activity when subjected to temperatures ranging from about 50°

C. to 105°

C.;

blending the sample, the at least two oligonucleotides of the first set, the at least two oligonucleotides of the second set, and the thermostable ligase to form an amplification mixture; and

subjecting the amplification mixture to a series of cycles comprising a denaturation treatment, wherein the ligated first oligonucleotide set is separated from the first nucleotide sequence and the litigated second oligonucleotide set is separated from the second nucleotide sequence, and a thermal hybridization treatment at a temperature of 50°

-85°

C., wherein the first oligonucleotide set hybridizes to the first nucleotide sequence and its oligonucleotides ligate to one another while the second oligonucleotide set hybridizes to the second nucleotide sequence and its oligonucleotides ligate to one another, to amplify exponentially the first and second nucleotide sequences in the DNA, wherein the at least two oligonucleotides of the second oligonucleotide set are complementary to the at least two oligonucleotides of the first oligonucleotide set with an oligonucleotide from the first oligonucleotide set complementing an oligonucleotide from the second oligonucleotide set with a single base overhang.

2. A method according to claim 1, wherein the single base overhang is a 3'"'"' end overhang.

3. A method for together amplifying nucleotide sequences which are complementary and together form separate strands of double stranded DNA molecules with one double-stranded DNA molecule having a genetic defect and another double-stranded DNA molecule having a normal form of the complementary nucleotide sequence, said method comprising:

providing a sample potentially containing a double-stranded DNA molecule with a nucleotide sequence having a genetic defect and a double-stranded DNA molecule with a normal form of the nucleotide sequence;

providing a first oligonucleotide set of at least two oligonucleotides suitable for ligation together at a first ligation junction and for hybridization without mismatch at the first ligation junction to the nucleotide sequence with the genetic defect, but not to the nucleotide sequence with the normal form, wherein the oligonucleotides of the first set hybridize adjacent to one another on the nucleotide sequence with the genetic defect and have a hybridization temperature of about 50°

C. to 85°

C.;

providing a second oligonucleotide set of at least two oligonucleotides suitable for ligation together at a second ligation junction and for hybridization without mismatch at the second ligation junction to the nucleotide sequence with the normal form, but not to the nucleotide sequence with the genetic defect, wherein the oligonucleotides of the second set hybridize adjacent to one another on the nucleotide sequence with the normal form and have a hybridization temperature of about 50°

C. to 85°

C.;

providing a third oligonucleotide set of at least two oligonucleotides suitable for ligation together at a third ligation junction and for hybridization without mismatch at the third ligation junction to a third nucleotide sequence, the third nucleotide sequence being complementary to the first nucleotide sequence and present in a second strand of the defective DNA molecule;

providing a fourth oligonucleotide set of at least two oligonucleotides suitable for ligation together at a fourth ligation junction and for hybridization without mismatch at the fourth ligation junction to a fourth nucleotide sequence, the fourth nucleotide sequence being complementary to the second nucleotide sequence and present in a second strand of the normal DNA molecule;

providing a thermostable ligase which does not become irreversibly denatured and lose its catalytic activity when subjected to temperatures ranging from about 50°

C. to 105°

C.;

blending the sample, the first set of oligonucleotides, the third set of oligonucleotides, and the thermostable ligase to form a first amplification mixture;

blending the sample, the second set of oligonucleotides, the fourth set of oligonucleotides, and the thermostable ligase to form a second amplification mixture; and

subjecting the first and second amplification mixtures to a series of cycles comprising a denaturation treatment, wherein the first and third oligonucleotide sets are separated from the nucleotide sequence with the genetic defect and the third nucleotide sequence, respectively, while the second and fourth oligonucleotide sets are separated from the nucleotide sequence with the normal form and the fourth nucleotide sequence, respectively, and a thermal hybridization treatment at a temperature of about 50°

C. to 85°

C., wherein the first oligonucleotide set hybridizes to the nucleotide sequence with the genetic defect, the second oligonucleotide set hybridizes to the nucleotide sequence with the normal form, the third oligonucleotide set hybridizes to the third nucleotide sequence, and the fourth oligonucleotide set hybridizes to the fourth nucleotide sequence, while the oligonucleotides of each set ligate to one another, to amplify exponentially the first, second, third, and fourth sequences, wherein the at least two oligonucleotides of the third oligonucleotide set are complementary to the at least two oligonucleotides of the first oligonucleotide set with an oligonucleotide from the first oligonucleotide set complementing an oligonucleotide from the third oligonucleotide set with a single base overhang and wherein the at least two oligonucleotides of the fourth oligonucleotide set are complementary to the at least two oligonucleotides of the second oligonucleotide set with an oligonucleotide from the second oligonucleotide set complementing an oligonucleotide from the fourth oligonucleotide set with a single base overhang.

4. A method according to claim 3, wherein each of the single base overhangs is a 3'"'"' end overhang.