Repair-mediated process for amplifying and detecting nucleic acid sequences

• US 6,004,826 A
• Filed: 10/27/1993
• Issued: 12/21/1999
• Est. Priority Date: 07/20/1988
• Status: Expired due to Term

• Current Assignee: David Segev
• Original Assignee: David Segev
• Inventors: Segev, David
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/155,938
• Time in Patent Office: 2,246 Days
• Field of Search: 435/6, 435/91.2, 435/91.52
• US Class Current: 438/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS

Abstract

This invention relates to a process for amplifying and detecting any desired specific nucleic acid sequence that exists in a nucleic acid or mixture thereof. The process comprises treating single strand RNA or separated complementary strands of DNA target with a molar excess of oligonucleotide complement pairs in which these oligonucleotide complement pairs have sequences complementary to the target, under hybridizing conditions. In one embodiment, the oligonucleotide complement pairs may have a gap of one or more bases which may be repaired (filled) by enzymes. The oligonucleotide complement pairs are joined together, forming joined, oligonucleotide product. The target/joined product hybrid nucleic acids are then denatured to single strands again, at which point both the target and the joined products can form hybrids with new oligonucleotide complement pairs. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired.

Claims

1. A process for detecting a specific nucleic acid target molecule in a sample by amplifying the nucleic acid target molecule, the process comprising the steps of:

a) treating the sample with at least two oligonucleotides for each strand of the target molecule, under hybridizing conditions,

i) wherein the oligonucleotides are selected so as to be sufficiently complementary to each strand of the target molecule to hybridize therewith under the hybridizing conditions; and

ii) wherein a gap of one or more bases is present between two oligonucleotides when the two oligonucleotides are hybridized to a strand of the target molecule; and

iii) wherein the oligonucleotides are selected so that the gaps between them will require less than all four types of bases to fill the gaps;

b) filling the gaps formed in step (a) with one or more bases complementary to the base or bases in the gaps and joining the base or bases filling the gaps to each other and to both adjacent hybridized oligonucleotides, thereby forming a joined oligonucleotide product;

c) treating the hybridized joined oligonucleotide product of step (b) under denaturing conditions to separate the joined oligonucleotide products from the target molecule to produce single-stranded molecules;

d) treating the single-stranded molecules produced in step (c) with an excess of at least two oligonucleotide complement pairs, under hybridizing conditions,

i) wherein each oligonucleotide complement pair comprises two oligonucleotides selected so as to be sufficiently complementary to each other, to each strand of the target molecule and to the joined oligonucleotide products to hybridize therewith under the hybridizing conditions; and

ii) wherein gaps of one or more bases are present between the oligonucleotides when the oligonucleotides are hybridized to each strand of the target molecule; and

iii) wherein the oligonucleotides are selected so that the gaps between them will require less than all four types of bases to fill the gaps;

e) filling in the gaps formed in step (d) with one or more bases complementary to the base or bases in the gaps and joining the bases filling the gaps to each other and to both adjacent hybridized oligonucleotides, thereby forming additional joined oligonucleotide products, resulting in the amplification of the target molecule; and

f) detecting the joined oligonucleotide products.

2. The process of claim 1, further including step

e'"'"') treating the hybridized oligonucleotides of step e) under denaturing conditions to separate the hybridized oligonucleotides and produce single-stranded molecules, wherein steps (d), (e) and (e'"'"') are repeated a desired number of times.

3. The process of claim 1, wherein the nucleic acid target molecule is double-stranded DNA and its strands are separated before or during step (a).

4. The process of claim 1, wherein the nucleic acid target molecule is single-stranded DNA.

5. The process of claim 1, wherein the nucleic acid target molecule is single-stranded RNA.

6. The process of claim 1, wherein the oligonucleotides are oligodeoxyribonucleotides.

7. The process of claim 1, wherein the oligonucleotide complement pairs are present as a molar excess in the range of 10⁵ to 10¹⁵ pairs per nucleic acid target molecule.

8. The process of claim 1, wherein the gaps are filled by a DNA polymerase and a DNA ligase.

9. The process of claim 8, wherein the DNA polymerase and the DNA ligase are immobilized on polymeric supports.

10. The process of claim 8, wherein the DNA polymerase is selected from the group consisting of E. coli DNA polymerase-I, Klenow fragments of E. coli DNA polymerase-I, T4 DNA polymerase, and reverse transcriptase and the DNA ligase is selected from the group consisting of E. coli DNA ligase and T4 DNA ligase.

11. The process of claim 8, wherein the DNA polymerase is heat stable.

12. The process of claim 8, wherein the DNA ligase is heat stable.

13. The process of claim 8, wherein the heat stable polymerase and ligase are isolated from a thermophilic bacteria.

14. The process of claim 1, wherein the oligonucleotides and/or deoxyribonucleic triphosphates are modified to be resistant to 3'"'"'→

5'"'"' exonuclease activity.

15. A process for detecting a specific nucleic acid target molecule in a sample by amplifying the nucleic acid target molecule, the process comprising the steps of:

a) treating the sample with at least two oligonucleotides for each strand of the target molecule, under hybridizing conditions,

i) wherein the oligonucleotides are selected so as to be sufficiently complementary to each strand of the target molecule to hybridize therewith under the hybridizing conditions; and

ii) wherein a gap of more than one bases is present between two oligonucleotides when the two oligonucleotides are hybridized to a strand of the target molecule; and

iii) wherein the oligonucleotides are selected so that the gaps between them will require less than all four types of bases to fill the gaps;

b) filling the gaps formed in step (a) with one or more bases complementary to the base or bases in the gaps and joining the base or bases filling the gaps to each other and to both adjacent hybridized oligonucleotides, thereby forming a joined oligonucleotide product;

c) treating the hybridized joined oligonucleotide product of step (b) under denaturing conditions to separate the joined oligonucleotide products from the target molecule to produce single-stranded molecules;

d) treating the single-stranded molecules produced in step (c) with an excess of at least two oligonucleotide complement pairs, under hybridizing conditions,

i) wherein each oligonucleotide complement pair comprises two oligonucleotides selected so as to be sufficiently complementary to each other, to each strand of the target molecule and to the joined oligonucleotide products to hybridize therewith under the hybridizing conditions; and

ii) wherein gaps of more than one bases are present between the oligonucleotides when the oligonucleotides are hybridized to each strand of the target molecule; and

iii) wherein the oligonucleotides are selected so that the gaps between them will require less than all four types of bases to fill the gaps;

e) filling in the gaps formed in step (d) with one or more bases complementary to the base or bases in the gaps and joining the bases filling the gaps to each other and to both adjacent hybridized oligonucleotides, thereby forming additional joined oligonucleotide products, resulting in the amplification of the target molecule; and

f) detecting the joined oligonucleotide products.