Multiplex method for nucleic acid detection

• US 6,653,078 B2
• Filed: 02/20/2001
• Issued: 11/25/2003
• Est. Priority Date: 03/13/1998
• Status: Expired due to Fees

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Wood, Keith V., Hartnett, James Robert, Gu, Trent, Mandrekar, Michelle, Rhodes, Richard Byron, Lewis, Martin K., Kephart, Daniel, Leippe, Donna, Shultz, John William, Andrews, Christine Ann, Welch, Roy
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): Chakrabarti, Arun K.
• Application Number:
  US09/788,847
• Publication Number:
  US 20030194699A1
• Time in Patent Office: 1,008 Days
• Field of Search: 435/6, 435/69.1, 435/214, 536/22.1, 536/23.1, 536/23.5, 536/29, 536/23, 536/24, 536/26, 536/27, 536/28
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Processes are disclosed using the depolymerization of a nucleic acid hybrid to qualitatively and quantitatively analyze for the presence of predetermined nucleic acid target sequences using a multiplex assay format. Applications of those processes include the detection of single nucleotide polymorphisms, identification of single base changes, speciation, genotyping, medical marker diagnostics, and the like.

Claims

1. A kit for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample comprising:

(A) a purified and isolated enzyme whose activity is to release one or more nucleotides from the 3′

terminus of a hybridized nucleic acid probe;

(B) a plurality of nucleic acid probes, each of said nucleic acid probes being complementary to a nucleic acid target sequence; and

(C) a nucleotide diphosphate kinase wherein said nucleoside diphosphate kinase is that encoded by Pyrococcus furiosis.

2. A kit for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample comprising:

(A) a purified and isolated enzyme whose activity is to release one or more nucleotides from the 3′

terminus of a hybridized nucleic acid probe;

(B) a plurality of nucleic acid probes, each of said nucleic acid probes being complementary to a nucleic acid target sequence;

(C) a nucleotide diphosphate kinase; and

(D) PRPP synthase.

3. A kit for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample comprising:

(A) a purified and isolated enzyme whose activity is to release one or more nucleotides from the 3′

terminus of a hybridized nucleic acid probe;

(B) a plurality of nucleic acid probes, each of said nucleic acid probes being complementary to a nucleic acid target sequence;

(C) a nucleotide diphosphate kinase; and

(D) ADP.

4. A composition of matter for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample comprising an aqueous solution that contains:

(A) a purified and isolated enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotide as a nucleoside triphosphate from hybridized nucleic acid probe;

(B) adenosine 5′

diphosphate;

(C) pyrophosphate;

(D) a purified and isolated nucleoside diphosphate kinase; and

(E) a plurality of nucleic acid probes, each of said nucleic acid probe being complementary to its respective predetermined nucleic acid target sequence.

5. The composition of matter according to claim 4, wherein said purified and isolated enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotides is a thermostable polymerase.

6. The composition of matter according to claim 5, wherein said purified and isolated nucleoside diphosphate kinase is that encoded by Pyrococcus furiosis.