Simultaneous sequencing of nucleic acids

• US 5,714,318 A
• Filed: 02/02/1994
• Issued: 02/03/1998
• Est. Priority Date: 06/02/1992
• Status: Expired due to Fees

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Sagner, Gregor, Kessler, Christoph, Blum, Helmut, Domdey, Horst
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/182,172
• Time in Patent Office: 1,462 Days
• Field of Search: 435/6, 435/172.3, 435/91.4, 435/91.41, 536/24.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

The invention concerns a new method for the simultaneous sequencing of nucleic acids using numerous double-stranded DNA adaptors.

Claims

1. A method for the simultaneous sequencing of nucleic acids, comprising:

(a) preparing numerous DNA fragments whose sequences are to be determined;

(b) dividing the DNA fragments to be sequenced into several groups;

(c) ligating double-stranded DNA adaptors to both ends of the divided DNA fragments from step (b) to produce DNA fragment/adaptor complexes, wherein the adaptors have a double-stranded region with a length of at least 5 nucleotides, have a first end that is compatible to the ends of the DNA fragments and a second end suitable for ligating into a vector, and wherein adaptors having different nucleotide sequences are ligated to the DNA fragments of each of the several groups;

(d) ligating DNA fragment/adaptor complexes from each of the several groups from step (c) into a base vector comprising a plurality of cleavage sites for a plurality of restriction endonucleases, which base vector has been cleaved with at least one of the restriction endonucleases, wherein the cleavage produces ends matching with the second end of one of the adaptors;

(e) selecting one vector from each of the several groups produced in step (d) and carrying out sequencing reactions on each of the ligated DNA fragment/adaptor complexes present in the selected vectors to produce sequencing products; and

(f) producing nucleotide sequence information by separating the sequencing products of step (e) from one another and thereafter identifying the separated sequencing products by hybridizing the separated sequencing products with at least one hybridization probe which comprises a DNA sequence which is complementary or identical to a 5 nucleotide portion of one adaptor of one single DNA fragment/adaptor complex.

2. The method as claimed in claim 1, wherein the at least one hybridization probe comprises a DNA sequence which is complementary to the DNA adaptor portion of the one single DNA fragment/adaptor complex.

3. The method as claimed in claim 1, wherein the DNA fragments of step (a) have blunt ends.

4. The method as claimed in claim 1, wherein the DNA fragments of step (a) have stepped ends.

5. The method as claimed in claim 1, wherein the second end of the double-stranded DNA adapters is stepped.

6. The method as claimed in claim 1, wherein the length of the double-stranded region of the double-stranded DNA adapters is 10 to 40 nucleotides.

7. The method as claimed in claim 1, wherein the double-stranded DNA adapters ligated to each end of the divided DNA fragments consist of the same nucleotide sequence.

8. The method as claimed in claim 1, wherein the double-stranded DNA adapters ligated to each end of the divided DNA fragments consist of different nucleotide sequences.

9. The method as claimed in claim 8, wherein the second ends of the double-stranded DNA adapters are stepped.

10. The method as claimed in claim 9, wherein the stepped second ends of the double-stranded DNA adapters ligated to each end of the divided DNA fragments are non-complementary.

11. The method as claimed in claim 1, wherein the base vector comprises an insertion region for inserting a DNA fragment/adaptor complex, the insertion region flanked on each side by at least one restriction endonuclease recognition sequence which contains at least one of (1) a sequence of at least 7 nucleotides and (2) a 5'"'"'-CG-3'"'"' nucleotide sequence.

12. The method as claimed in claim 11, wherein the at least one restriction endonuclease recognition sequence is selected from the group consisting of NotI, SfiI, RsrII, SgrAI, SwaI, PacI, AscI, PmeI, Sse83871, SrsI and I-SceI.

13. The method as claimed in claim 1, wherein each of the plurality of cleavage sites is flanked by transcription terminators.

14. The method as claimed in claim 1, wherein the at least one hybridization probes is non-radioactively labelled.

15. The method as claimed in claim 14, wherein the at least one hybridization probe is non-radioactively labelled with a label independently selected from the group consisting of biotin, digoxigenin, a fluorescent label, a luminescent label, and an enzyme.

16. The method as claimed in claim 1, wherein step (f) comprises the following steps:

(f1) separating the sequencing products of step (e) according to the size thereof;

(f2) transferring the separated sequencing products from step (f1) onto a suitable carrier for binding nucleic acids;

(f3) reversibly hybridizing the separated sequencing products from step (f2) with at least one hybridization probe which comprises a DNA sequence which is complementary to one single DNA fragment/adaptor complex; and

(f4) producing nucleotide sequence information by analyzing the hybridization of the separated sequencing products from step (f3).

17. The method as claimed in claim 16, wherein the at least one hybridization probe comprises a DNA sequence which is complementary to the DNA adaptor portion of the one single DNA fragment/adaptor complex.

18. The method as claimed in claim 16, wherein step (f2) further comprises immobilizing the transferred separated sequencing products on the carrier.

19. The method as claimed in claim 16, further comprising, after step (f4), removing the hybridized at least one hybridization probe from the carrier, and thereafter repeating steps (f3) and (f4) using a different hybridization probe which comprises a DNA sequence which is complementary to a different single DNA fragment/adaptor complex.

20. A method for converting an already existing gene bank into a modified gene bank on which a simultaneous sequencing of nucleic acids can be carried out, wherein the already existing gene bank comprises a plurality of different DNA fragments which have been ligated into a base vector at an insertion region thereof, wherein the insertion region is flanked on a side by at least one restriction endonuclease recognition sequence which contains at least one of (1) a sequence of at least 7 nucleotides and (2) a 5'"'"'-CG-3'"'"' nucleotide sequence, the method comprising:

(a) cleaving the gene bank with an enzyme at the at least one restriction endonuclease recognition sequence to produce numerous DNA fragments;

(b) dividing the DNA fragments into several groups; and

(c) ligating the divided DNA fragments from step (b) with a double-stranded DNA adaptor to produce ligation products, wherein the adaptor has a double-stranded region with a length of at least 5 nucleotides and ends on both sides which are suitable for ligation with the at least one restriction endonuclease recognition sequence, and wherein an adaptor having a different nucleotide sequence is ligated to a DNA fragment from each of the several groups.

21. The method as claimed in claim 20, further comprising, after step (c),

(d) separating the ligation products from by-products.

22. The method as claimed in claim 20, wherein the double-stranded DNA adaptor has a sequence which is selected such that the at least one restriction endonuclease recognition sequence is eliminated by ligation of the double-stranded DNA adaptor in step (c).

23. The method as claimed in claim 21, wherein step (d) comprises recleaving the ligation products with the enzyme used in step (a).

24. The method as claimed in claim 20, wherein the at least one restriction endonuclease recognition sequence is selected from the group consisting of NotI, SfiI, RsrII, SgrAI, SwaI, PacI, AscI, PmeI, Sse83871, SrsI and IsceI.

25. The method as claimed in claim 20, wherein the adaptor molecule has a symmetrical sequence.

26. The method as claimed in claim 20, wherein the adaptor molecule has an unsymmetrical sequence.

27. The method as claimed in claim 20, wherein the insertion region is flanked on each side by different restriction endonuclease recognition sequences each containing at least one of (1) a sequence of at least 7 nucleotides and (2) a 5'"'"'-CG-3'"'"' nucleotide sequence, and wherein the steps (a)-(c) are carried out for each at least one restriction endonuclease recognition sequence.