Method, compositions and kit for detection of microorganisms and bi-directional sequencing of nucleic acid polymers

• US 20030082535A1
• Filed: 03/07/2001
• Published: 05/01/2003
• Est. Priority Date: 05/01/1996
• Status: Abandoned Application

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Inventors: Hui, May, Dunn, James M., Lacroix, Jean-Michel, Leushner, James
• Application Number:
  US09/802,110
• Publication Number:
  US 20030082535A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks

Abstract

Evaluation of a sample for the presence and qualitative nature of a microorganism can be performed in a single vessel by combining a natural abundance DNA sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as ThermoSequenase™ which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide triphosphate feedstocks, and a chain terminating nucleotide triphosphate. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.

Claims

1. A composition comprising a mixture of four deoxynucleotide triphosphates and at least one dideoxynucleotide triphosphate corresponding to one of the four deoxynucleotide triphosphates, wherein the dideoxynucleotide triphosphate is present in a mole ratio to the corresponding deoxynucleotide triphosphate of from 1:

50 to 1;

500, said composition further comprising a thermally stable polymerase enzyme which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than 0.4 times the rate of incorporation of deoxynucleotides.

2. The composition according to claim 1, wherein the mole ratio is from 1:

100 to 1;

300.

3. A kit for detection of a target microorganism comprising, in packaged combination,

(a) a pair of primers which bind to the sense and antisense strands, respectively, and flank a selected region within the genome target microorganism;

and

(b) a mixture of four deoxynucleotide triphosphates and at least dideoxynucleotide triphosphate corresponding to one of the four deoxynucleotide triphosphates, wherein the dideoxynucleotide triphosphate is present in a mole ratio to the corresponding deoxynucleotide triphosphate of from 1;

50 to 1;

1000

(c) a polymerase enzyme which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than 0.4 times the rate of incorporation of deoxynucleotides.

4. The kit according to claim 3, wherein the mole ratio is from 1:

100 to 1;

500.

5. The kit according to claim 3, wherein at least one of the primers is labeled with a fluorescent label.

6. The kit according to claim 3, wherein the primers are each labeled with a spectroscopically-distinct fluorescent label.

7. The kit according to claim 3, wherein the target microorganism is Chlamydia trachomatis.

8. The kit according to claim 7, wherein the first and second primers are selected from the group consisting of the oligonucleotides given by Seq. ID. Nos. 1-17.

9. The kit according to claim 3, wherein the target microorganism is human immunodeficiency virus.

10. The kit according to claim 9, wherein the first and second primers are selected from the group consisting of the oligonucleotides given by Seq. ID. Nos. 18-20.

11. The kit according to claim 3, wherein the target microorganism is human papilloma virus.

12. The kit according to claim 11, wherein the first and second primers are selected from the group consisting of the oligonucleotides given by Seq. ID. Nos. 21-22.