Oligonucleotide primer composition

Patent metadata

Field	Value
Publication number	US-9523133-B2
Application number	US-201213678317-A
Country	US
Kind code	B2
Filing date	Nov 15, 2012
Priority date	Sep 30, 2004
Publication date	Dec 20, 2016
Grant date	Dec 20, 2016

Abstract

Oligonucleotide primer useful for synthesizing a cDNA copy of HIV-1 nucleic acids from a broad range of HIV-1 subtypes, including M group and O group variants.

First claim

What is claimed is:

1. A composition comprising an oligonucleotide primer,
wherein the base sequence of the oligonucleotide primer consists of a target-hybridizing sequence and an upstream phage T7 RNA polymerase promoter sequence,
wherein the base sequence of the upstream phage T7 RNA polymerase promoter sequence is the base sequence of SEQ ID NO:30,
wherein the base sequence of the target-hybridizing sequence is the base sequence of SEQ ID NO:15, and
wherein the composition further comprises a hybridization probe, and wherein the base sequence of the hybridization probe is the base sequence of the molecular torch of SEQ ID NO:24.

2. A composition comprising an oligonucleotide primer,
wherein the base sequence of the oligonucleotide primer consists of a target-hybridizing sequence and an upstream phage T7 RNA polymerase promoter sequence,
wherein the base sequence of the upstream phage T7 RNA polymerase promoter sequence is the base sequence of SEQ ID NO:30,
wherein the base sequence of the target-hybridizing sequence is the base sequence of SEQ ID NO:15, and
wherein the composition further comprises a hybridization probe having a target-hybridizing sequence, a fluorophore moiety and a quencher moiety, and wherein the base sequence of the target-hybridizing sequence is the base sequence of SEQ ID NO:23.

3. A method of synthesizing a cDNA strand complementary to an HIV-1 nucleic acid contained in a test sample, the method comprising the steps of:
(a) contacting the HIV-1 nucleic acid with a composition comprising an oligonucleotide primer in the presence of a reverse transcriptase enzyme and deoxyribonucleotide triphosphates,
wherein the base sequence of the oligonucleotide primer consists of a target-hybridizing sequence and an upstream phage T7 RNA polymerase promoter sequence,
wherein the base sequence of the upstream phage T7 RNA polymerase promoter sequence is the base sequence of SEQ ID NO:30, and
wherein the base sequence of the target-hybridizing sequence is the base sequence of SEQ ID NO:15;

(b) hybridizing the oligonucleotide primer to the HIV-1 nucleic acid, whereby a heretoduplex is formed;
(c) permitting extension of the oligonucleotide primer of the heteroduplex by the reverse transcriptase enzyme using the HIV-1 nucleic acid as the template, thereby synthesizing the cDNA strand; and
(d) determining with a hybridization probe that the cDNA strand was synthesized in step (c),
wherein the hybridization probe is the molecular torch of SEQ ID NO:24.

4. A method of synthesizing a cDNA strand complementary to an HIV-1 nucleic acid contained in a test sample, the method comprising the steps of:
(a) contacting the HIV-1 nucleic acid with a composition comprising an oligonucleotide primer in the presence of a reverse transcriptase enzyme and deoxyribonucleotide triphosphates,
wherein the base sequence of the oligonucleotide primer consists of a target-hybridizing sequence and an upstream phage T7 RNA polymerase promoter sequence,
wherein the base sequence of the upstream phage T7 RNA polymerase promoter sequence is the base sequence of SEQ ID NO:30, and
wherein the base sequence of the target-hybridizing sequence is the base sequence of SEQ ID NO:15;

(b) hybridizing the oligonucleotide primer to the HIV-1 nucleic acid, whereby a heretoduplex is formed;
(c) permitting extension of the oligonucleotide primer of the heteroduplex by the reverse transcriptase enzyme using the HIV-1 nucleic acid as the template, thereby synthesizing the cDNA strand; and
(d) determining with a hybridization probe that the cDNA strand was synthesized in step (c),
wherein the hybridization probe comprises a fluorophore moiety and a quencher moiety, and wherein step (d) comprises detecting an optical signal, and
wherein the target-hybridizing sequence of the hybridization probe is SEQ ID NO:23.

5. A composition comprising:
an oligonucleotide primer, wherein the base sequence of the oligonucleotide primer consists of a target-hybridizing sequence and an upstream RNA polymerase promoter sequence, wherein the base sequence of the upstream RNA polymerase promoter sequence is the base sequence of SEQ ID NO:30, and wherein the base sequence of the target-hybridizing sequence is the base sequence of SEQ ID NO:15; and
a hybridization probe having a fluorophore moiety, a quencher moiety, and the base sequence of SEQ ID NO:23.

6. The composition of claim 5 , wherein the hybridization probe is the molecular torch hybridization probe of SEQ ID NO:24.

7. A composition comprising an oligonucleotide primer, wherein the base sequence of the oligonucleotide primer is SEQ ID NO:19.

8. The composition of claim 4 , further comprising a reverse transcriptase enzyme and deoxyribonucleotide triphosphates.

9. The composition of claim 8 , wherein the reverse transcriptase enzyme is MMLV reverse-transcriptase.

10. The composition of claim 8 , further comprising ribonucleotide triphosphates.

11. The composition of claim 10 , further comprising T7 RNA polymerase.

12. The method of either claim 3 or claim 4 , further comprising a step, taking place before step (a), of isolating the HIV-1 nucleic acid from the test sample.

13. The method of claim 12 , wherein the test sample is a blood sample.

Assignees

• Gen Probe Inc

Inventors

• Schroder Astrid R W

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q1/6865

  Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS] · CPC title

• C12Q1/703 Primary

  Viruses associated with AIDS · CPC title

• C12Q2545/114

  involving a quantitation step · CPC title