Inverse linkage oligonucleotides for chemical and enzymatic processes

• US 5,462,854 A
• Filed: 04/19/1993
• Issued: 10/31/1995
• Est. Priority Date: 04/19/1993
• Status: Expired due to Fees

• Current Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Original Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Inventors: Cook, Ronald M., Coassin, Peter J., Rampal, Jang B., Konrad, Kenneth D.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/050,681
• Time in Patent Office: 925 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 935/78
• US Class Current: 435/6.12
• 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: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]

Abstract

Inverse Linkage Oligonucleotides ("ILO") useful in enzymatic process are disclosed. Particularly preferred ILOs are amenable to enzymatic elongation from either, or most preferably, both termini. In a particularly preferred embodiment, each terminus of an ILO has an enzymatically functional 3'"'"' group. Accordingly, under appropriate conditions and in the presence of, e.g., dNTPs, enzyme, sample DNA, and ILO comprising a first region complementary to a first region of the sample DNA and a second region complementary to a second, different region of the sample DNA, exponential amplification of the sample DNA can be effectuated.

Claims

1. A process for the amplification of a double-stranded nucleic acid molecule, said molecule comprising a strand and a complementary strand, comprising the steps of:

(a) separating the strand from the complementary strand;

(b) treating the strand and the complementary strand to at least one 3'"'"'-Inverse Linkage Oligonucleotide, where each 3'"'"'-Inverse Linkage Oligonucleotide comprises a first sequence portion complementary to a region on said strand and a second sequence portion complementary to a region on said complementary strand, where said regions are not complementary with each other, under conditions sufficient to permit the first sequence portion of said 3'"'"'-Inverse Linkage Oligonucleotide to hybridize to said region on said strand or the second sequence portion of said 3'"'"'-Inverse Linkage Oligonucleotide to hybridize to said region on said complementary strand;

(c) extending said hybridized 3'"'"'-Inverse Linkage Oligonucleotide with a polymerase along said strand or said complementary strand, respectively, to form 3'"'"'-Inverse Linkage Oligonucleotide products, wherein each of said products includes a segment capable of hybridizing to the portion of the 3'"'"'-Inverse Linkage Oligonucleotide not hybridized in step (b);

(d) separating 3'"'"'-Inverse Linkage Oligonucleotide products from the strand and the complementary strands to form single-stranded products, wherein at least one 3'"'"'-Inverse Linkage Oligonucleotide product has a first 3'"'"'-termini complementary to a second 3'"'"'-termini, said first 3'"'"'-termini capable of hybridizing to said second 3'"'"'-termini to form a partially double-stranded circle;

(e) treating the single-stranded products of step (d) with the 3'"'"'-Inverse Linkage Oligonucleotide of step (b) and allowing said first and second 3'"'"'-termini to hybridize to form said partially double-stranded circle; and

(f) extending the 3'"'"'-Inverse Linkage Oligonucleotide and the appropriate 3'"'"'-termini of said circle with a polymerase.

2. The process of claim 1 wherein said 3'"'"'-Inverse Linkage Oligonucleotide comprises a label.

3. The process of claim 2 wherein said label is located at an inverse linkage of said 3+-Inverse Linkage Oligonucleotide.

4. The process of claim 1 wherein the length of said 3'"'"'-Inverse Linkage Oligonucleotide is between about 8 and about 100 nucleotides.

5. The process of claim 1 wherein said macromolecule is human genomic DNA.

6. The process of claim 1 wherein said 3'"'"'-Inverse Linkage Oligonucleotide products are selected from the group consisting of 3'"'"'-Inverse Linkage Oligonucleotide Amplicons, Multimeric 3'"'"'-Inverse Linkage Oligonucleotide Amplicons, and Circular Double-Stranded 3'"'"'-Inverse Linkage Oligonucleotide Amplicons.

7. The process of claim 6 wherein said 3'"'"'-Inverse Linkage Oligonucleotide Amplicons are selected from the group consisting of Partially Extended 3'"'"'-Inverse Linkage Oligonucleotide Amplicons and Fully Extended 3'"'"'-Inverse Linkage Oligonucleotides Amplicons.

8. An ILO amplification product obtained by the process of claim 1, said product comprising circular double stranded ILO amplicons.

9. A process for detecting the presence of a double-stranded nucleic acid molecule, said molecule comprising a strand and a complementary strand, comprising the steps of:

(a) separating the strand from the complementary strand;

(b) treating the strand and the complementary strand to at least one immobilized 3'"'"'-Inverse Linkage Oligonucleotide, wherein said 3'"'"'-Inverse Linkage Oligonucleotide comprises at least one inverse linkage, said 3'"'"'-Inverse Linkage Oligonucleotide being immobilized to a solid support, where each 3'"'"'-Inverse Linkage Oligonucleotide comprises a first sequence portion complementary to a region on said strand and a second sequence portion complementary to a region on said complementary strand, where said regions are not complementary to each other, under conditions sufficient to permit said 3'"'"'-Inverse Linkage Oligonucleotide to hybridize to hybridization member selected from the group consisting of;

(i) said strand,

(ii) said complementary strand, and

(iii) said strand and said complementary strand;

(c) extending said 3'"'"'-Inverse Linkage Oligonucleotide with a polymerase along the region of hybridization to form extended immobilized 3'"'"'-Inverse Linkage Oligonucleotides, each of which includes a segment capable of hybridizing to the portion of the 3'"'"'-Inverse Linkage Oligonucleotide not hybridized in step (b);

(d) separating said hybridization member from said extended immobilized 3'"'"'-Inverse Linkage Oligonucleotide;

(e) detecting said extended immobilized 3'"'"'-Inverse Linkage Oligonucleotide as an indication of the presence of said molecule.

10. The process of claim 9 wherein the presence of a specific sequence is detected on said extended, immobilized 3'"'"'-Inverse Linkage Oligonucleotide said specific sequence being different than said first sequence portion complementary to a region on said strand and a second sequence portion complementary to a region on said complementary strand of said 3'"'"'-Inverse Linkage Oligonucleotide.

11. The process of claim 9 wherein said detection is by means of an allele specific oligonucleotide (ASO).

12. The process of claim 11 wherein said ASO is labelled.