Detection of nucleic acid hybrids

• US 6,730,479 B2
• Filed: 02/22/2001
• Issued: 05/04/2004
• Est. Priority Date: 03/13/1998
• Status: Expired due to Fees

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Olson, Ryan J., Hartnett, James Robert, Gu, Trent, Mandrekar, Michelle, Rhodes, Richard Byron, Lewis, Martin K., Kephart, Daniel, Leippe, Donna, Andrews, Christine Ann, Welch, Roy, Wood, Keith V., Shultz, John William
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/790,417
• Publication Number:
  US 20010031470A1
• Time in Patent Office: 1,167 Days
• Field of Search: 435/6, 435/320.1, 435/252.8, 435/174, 435/183, 536/23.7, 530/350, 935/10, 935/24, 935/72
• 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 a predetermined nucleic acid. Applications of those processes include the detection of single nucleotide polymorphisms, identification of single base changes, speciation, determination of viral load, genotyping, medical marker diagnostics, and the like.

Claims

1. A method of detecting the presence of nuclease activity in a sample comprising:

a) providing a mixture comprising

i) a solution suspected of containing nuclease activity; and

ii) a deoxyribonucleic acid substrate, wherein said nucleic acid substrate is selected from the group consisting of linear deoxyribonucleic acid and closed circular deoxyribonucleic acid;

b) incubating said mixture for a period of time sufficient for said nuclease to act on said nucleic acid;

c) reacting said mixture under conditions such that free nucleotides are produced;

wherein said reacting step further comprises;

i) providing a second solution comprising said nucleic acid substrate, adenosine 5′

diphosphate, and pyrophosphate, said nucleic acid substrate having a terminal nucleotide and terminal internucleotide phosphodiester bond, said terminal nucleotide covalently joined to said nucleic acid by said terminal internucleotide phosphodiester bond;

ii) depolymerizing said nucleic acid at a terminal nucleotide by enzymatically cleaving said terminal internucleotide phosphodiester bond to form a free nucleoside triphosphate molecule with a pyrophosphate molecule according to the reaction;

 

dNA_n+PP_i→

dNA<sub>n−

1</sub>+dNTP

iii) enzymatically transferring terminal 5′

phosphate groups from the nucleoside triphosphate molecules to adenosine 5′

-diphosphate molecules to form adenosine 5′

-triphosphate according to the following general reaction;

2. The method of claim 1 wherein said detecting step further comprises quantitating said adenosine 5′

triphosphate.

3. The method of claim 1 wherein the detecting step is selected from the group consisting of luciferase and NADH detection systems.

4. The method of claim 1, wherein said depolymerization is catalyzed by E. coli polymerase 1.

5. The method of claim 1, wherein said depolymerization is catalyzed by T4 DNA polymerase.

6. A method of detecting the presence of exonuclease activity comprising:

a) providing a mixture comprising

i) a solution suspected of containing exonuclease activity; and

ii) a deoxyribonucleic acid substrate;

b) incubating said mixture to allow degradation of said deoxyribonucleic acid substrate by said exonuclease activity, thereby forming nucleoside monophosphates;

c) converting said nucleoside monophosphates into nucleoside triphosphates

i) providing a second solution comprising phosphoribosylpyrophosphate, adenosine 5′

diphosphate, and phosphoribosyl transferase;

ii) enzymatically transferring pyrophosphate from phophoribosylpyrophosphate molecules to deoxyadenosine monophosphate molecules to form deoxyadenosine triphosphate molecules according to the following reaction catalyzed by phosphoribosyl pyrophosphate transferase;

7. The method of claim 6 wherein said detecting step further comprises quantitating said adenosine 5′

triphosphate.

8. The method of claim 6 wherein the detecting step is selected from the group consisting of luciferase and NADH detection systems.