Direct bacterial lysate sequencing

• US 6,605,434 B1
• Filed: 03/16/2000
• Issued: 08/12/2003
• Est. Priority Date: 03/16/1999
• Status: Expired due to Fees

• Current Assignee: Human Genome Sciences Incorporated (GSK plc)
• Original Assignee: Human Genome Sciences Incorporated (GSK plc)
• Inventors: Ferrie, Andrew, Zhong, Haibing
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/526,731
• Time in Patent Office: 1,244 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 435/968, 536/25.32, 422/68.1, 204/461
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The present invention relates to novel methods for sequencing nucleic acid molecules. More specifically, methods are provided for sequencing nucleic acid molecules present in bacterial lysates without the need to purify nucleic acid templates from highly soluble cellular components.

Claims

1. A method for sequencing a nucleic acid template comprising:

(a) incubating bacterial host cells containing the nucleic acid template at about 95°

C. to about 96°

C. for about 20 to about 25 minutes to produce a cell lysate; and

(b) sequencing the nucleic acid template in the cell lysate using a sequencing reaction and one or more detectable labels to detect the products of the sequencing reaction, wherein the nucleic acid template is not purified from highly soluble cellular components released from the bacterial host cells upon lysis and is not amplified by polymerase chain reaction.

2. The method of claim 1 comprising washing the bacterial host cell with water prior to lysing.

3. The method of claim 2 comprising lysing the bacterial host cell in water.

4. The method of claim 1 wherein the cell lysate is incubated between about 4 and about 15°

C. prior to sequencing.

5. The method of claim 4 wherein the cell lysate is centrifuged prior to sequencing.

6. The method of claim 5 wherein the cell lysate is incubated for about 5 minutes prior to centrifugation.

7. The method of claim 6 wherein the bacterial host cell is Escherichia coli.

8. The method of claim 7 comprising culturing the bacterial host cell in a broth selected from the group consisting of:

(a) Luria broth; and

(b) Terrific broth,

prior to lysing.

9. The method of claim 1 wherein the detectable labels comprise one or more fluorescent dyes.

10. The method of claim 9 wherein the fluorescent dyes are d-rhodamine dyes having structural formulas shown in (FIG. 6A), (FIG. 6B), (FIG. 6C), (FIG. 6D), (FIG. 6E) or (FIG. 6F).

11. The method of claim 9 wherein the fluorescent dyes are d-rhodamine dyes having structural formulas shown in (FIG. 7A), (FIG. 7B), (FIG. 7C) or (FIG. 7D).

12. The method of claim 9 wherein the fluorescent dyes are fluorescein/d-rhodamine dyes having structural formulas shown in (FIG. 8A), (FIG. 8B), (FIG. 8C) or (FIG. 8D).

13. The method of claim 12 wherein the fluorescent dyes are fluorescein/d-rhodamine dyes having structural formulas shown in (FIG. 9A), (FIG. 9B), (FIG. 9C) or (FIG. 9D).

14. The method of claim 1 wherein the detectable labels comprise infrared labels.

15. A method for sequencing a nucleic acid template comprising:

(a) incubating bacterial host cells containing the nucleic acid template at about 95°

C. to about 96°

C. for about 20 to about 25 minutes to produce a cell lysate; and

(b) forming in the cell lysate a mixture of a first, a second, a third, and a fourth class of polynucleotides such that;

(i) the polynucleotides in the first class include a 3′

-terminal dideoxyadenosine and are labeled with a first detectable label;

(ii) the polynucleotides in the second class include a 3′

-terminal dideoxycytidine and are labeled with a second detectable label;

(iii) the polynucleotides in the third class include a 3′

-terminal dideoxyguanosine and are labeled with a third detectable label;

(iv) the polynucleotides in the fourth class include a 3′

-terminal dideoxythymidine and are labeled with a fourth detectable label;

wherein the nucleic acid template is not purified from highly soluble cellular components released from the bacterial host cells upon lysis and is not amplified by polymerase chain reaction.

16. The method of claim 15 wherein the detectable labels comprise one or more fluorescent dyes.

17. The method of claim 16 wherein the fluorescent dyes are d-rhodamine dyes having structural formulas shown in (FIG. 6A), (FIG. 6B), (FIG. 6C), (FIG. 6D), (FIG. 6E) or (FIG. 6F).

18. The method of claim 16 wherein the fluorescent dyes are d-rhodamine dyes having structural formulas shown in (FIG. 7A), (FIG. 7B), (FIG. 7C) or (FIG. 7D).

19. A method of sequencing a nucleic acid template comprising:

(a) hybridizing a primer to the nucleic acid template;

(b) forming a mixture comprising the nucleic acid template, deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate, a DNA polymerase, and a dideocynucleotide;

(c) incubating the mixture of (b) under conditions sufficient to synthesize a population of DNA molecules complementary to a portion of the nucleic acid template; and

(d) separating said synthesized DNA molecules by size so that at least a part of the nucleotide sequence of the nucleic acid template can be determined;

wherein;

(a) the nucleic acid template is present in a cell lysate prepared by incubating bacterial host cells which contain the nucleic acid template at about 95°

C. to about 96°

C. for about 20 to about 25 minutes;

(b) the nucleic acid template is not purified from highly soluble cellular components released from the bacterial host cells upon lysis and is not amplidied by polymerase chain reaction; and

(c) the members of the population of DNA molecules are labeled with a detectable label.

20. The method of claim 19 wherein the detectable label comprises one or more fluorescent dyes.

21. The method of claim 20 wherein the fluorescent dyes are d-rhodamine dyes having structural formulas shown in (FIG. 6A), (FIG. 6B), (FIG. 6C), (FIG. 6D), (FIG. 6E) or (FIG. 6F).

22. The method of claim 20 wherein the fluorescent dyes are d-rhodamine dyes having structural formulas shown in (FIG. 7A), (FIG. 7B), (FIG. 7C) or (FIG. 7D).

23. The method of claim 20 wherein the fluorescent dyes are fluorescein/d-rhodamine dyes having structural formulas shown in (FIG. 8A), (FIG. 8B), (FIG. 8C) or (FIG. 8D).

24. The method of claim 23 wherein the fluorescent dyes are fluorescein/d-rhodamine dyes having structural formulas shown in (FIG. 9A), (FIG. 9B), (FIG. 9C) or (FIG. 9D).

25. The method of claim 19 wherein the detectable labels comprise one or more infrared labels.

26. A method for high-throughput sequencing of nucleic acid templates comprising:

(a) culturing more than one bacterial host cell such that host cells containing the same nucleic acid templates are separated from host cells containing different nucleic acid templates;

(b) lysing the cells of at least one bacterial host cell by incubating said cells at about 95°

C. to about 96°

C. for about 20 to about 25 minutes to produce a cell lysate; and

(c) sequencing the nucleic acid template in the cell lysate using a sequencing reaction and one or more detectable labels to detect the products of the sequencing reaction, wherein;

(a) the detectable labels comprise fluorescent dyes;

(b) the nucleic acid template is not purified from highly soluble cellular components released from the bacterial host cells upon lysis; and

(c) the nucleic acid template is not amplified by polymerase chain reaction.

27. The method of claim 26 wherein the fluorescent dyes are d-rhodamine dyes having structural formulas shown in (FIG. 6A), (FIG. 6B), (FIG. 6C), (FIG. 6D), (FIG. 6E) or (FIG. 6F).

28. The method of claim 26 wherein the fluorescent dyes are d-rhodamine dyes having structural formulas shown in (FIG. 7A), (FIG. 7B), (FIG. 7C) or (FIG. 7D).

29. The method of claim 26 wherein the fluorescent dyes are fluorescein/d-rhodamine dyes having structural formulas shown in (FIG. 8A), (FIG. 8B), (FIG. 8C) or (FIG. 8D).

30. The method of claim 29 wherein the fluorescent dyes are fluorescein/d-rhodamine dyes having structural formulas shown in (FIG. 9A), (FIG. 9B), (FIG. 9C) or (FIG. 9D).

31. The method of claim 26 comprising culturing more than one bacterial host cell on a solid culture medium to produce colonies.

32. The method of claim 31 comprising the transfer of cells of the colonies to a liquid culture medium for culturing.