Linear amplification mediated PCR (LAM PCR)

• US 6,514,706 B1
• Filed: 07/16/2001
• Issued: 02/04/2003
• Est. Priority Date: 10/26/1998
• Status: Expired due to Fees

• Current Assignee: Christoph Von Kalle, Manfred Schmidt
• Original Assignee: Christoph Von Kalle, Manfred Schmidt
• Inventors: Schmidt, Manfred, Von Kalle, Christoph
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/830,337
• Time in Patent Office: 568 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

A high sensitive method for identifying and/or sequencing an unknown DNA or RNA sequence flanking a known DNA or RNA region is described, wherein,

(a) one or more DNA or RNA fragments are subjected in a first step to one or more linear PCR steps using one more primers,

(b) the single strands obtained are complemented to form double strands,

(c) the double strands are digested by one or more restriction enzymes in order to produce smooth and/or cohesive ends,

(d) an oligonucleotide of known sequence is added at the digested ends, and

(e) the thus obtained DNA fragments are amplified and detected.

Claims

1. A method for identifying and/or sequencing an unknown or target DNA or RNA sequence flanking a known DNA or RNA region, the method comprising:

(a) subjecting one or more nucleotide sequences in a first step to one or more linear PCR steps using one or more primers to form single strands,

(b) complementing or pairing the single strands obtained from step (a) with arbitrary or non-specific priming sequences to form double strands,

(c) digesting the double strands with one or more restriction enzymes in order to produce smooth and/or cohesive ends,

(d) attaching, annealing or ligating an oligonucleotide of known sequence to the digested ends, and

(e) amplifying and detecting the modified DNA fragments.

6. The method according to claim 1, wherein hexanucleotide primers are used in step (b) to construct the complementary DNA strand.

7. The method according to claim 1, wherein degenerated primers are used in step (b) to construct the complementary DNA strand.

8. The method according to claim 1, the DNA fragments obtained in step (a) is complemented in a solid phase to form double strands.

9. The method according to claim 1, wherein step (c) is carried out in a solid phase.

10. The method according to claim 1, wherein steps 1(d) is carried out in a solid phase and the target DNA is then immediately amplified using a solid phase ligation mediated PCR technique.

11. The method according to claim 1, wherein the oligonucleotide in step 1l(d) is a linker cassette.

12. The method according to claim 1, wherein poly (A, T, G or C) tailing is performed in step 1(d).

13. The method according to claim 11, wherein the target DNA is denatured after step 1(d) and amplified using a PCR technique, the PCR technique being carried out using a primer which is complementary to the linker and a second primer which is complementary to the known DNA region.

14. The method according to claim 13, wherein the target DNA obtained by the first PCR technique is subjected to a second amplification using a nested PCR technique.

15. The method according to claim 13 or 14, wherein the target DNA is detected by gel electrophoresis, blotting or sequencing, after amplification.

16. The method according to claim 1, wherein step (c) is carried out that digestion does not occur within the known part of the target DNA sequence.

17. The method according to claim 16, wherein an oligonucleotide is added only at the digested end.

18. The method according to claim 1 or 2, wherein the genomic localization of transposons, retroviral integration events, retroviral vectors, transcriptionally active regions, resistance genes and/or transgenes is detected, amplified, identified and/or sequenced.

2. A method for identifying and/or sequencing an unknown or target DNA or RNA flanking a known DNA or RNA sequence, the method comprising:

(a) subjecting one or more DNA or RNA fragments in a first step to one or more linear PCR steps to form single strands, using one or more primers, the primer(s) being provided with a partner of a specific binding pair, said specific binding pair having a first binding partner and a second binding partner,

(b) separating the single strand fragments carrying the first binding partner from the reaction mixture by means of the second binding partner of the specific binding pair,

(c) complementing or pairing the single strands obtained in step (a) with arbitrary or non-specific priming sequences to form double strands,

(d) digesting the double strands with one or more restriction enzymes in order to produce smooth and/or cohesive ends,

(e) attaching, annealing or ligating an oligonucleotide of known sequence to the digested ends, and

(f) amplifying and detecting the modified DNA fragments.

3. The method according to claim 2, wherein the specific binding pair is selected from the group consisting of biotin and avidin and biotin and streptavidin.

4. The method according to claim 2, wherein one or more primers labelled with biotin are used in step (a) and it (they) is (are) immediately thereafter separated from the reaction mixture by specific binding to avidin or streptavidin.

5. The method according to claim 2, wherein the second partner of the specific bind pair is immobilized on beads or in a coated flask.

19. The method according to claim 2, wherein hexanucleotide primers are used in step 2(c) to construct the complementary DNA strand.

20. The method according to claim 2, wherein degenerated primers are used in step 2(c) to construct the complementary DNA strand.

21. The method according to claim 2, wherein the DNA fragments from step 2(b) are complemented in a solid phase to form double strands.

22. The method according to claim 2, wherein step 2(d) is carried out in a solid phase.

23. The method according to claim 2, wherein step 2(e) is carried out in a solid phase and the target DNA is immediately amplified using a solid phase ligation mediated PCR technique.

24. The method according to claim 2, wherein the oligonucleotide in step 2(e) is a linker cassette.

25. The method according to claim 2, wherein poly (A, T.

G or C) tailing is performed in step 2(e).

26. The method according to claim 24, wherein the target DNA is denatured after step 2(e) and amplified using a PCR technique, the PCR technique being carried out using a primer which is complementary to the linker and a second primer which is complementary to the known DNA region.

27. The method according to claim 26, wherein the target DNA obtained by the first PCR technique is subjected to a second amplification using a nested PCR technique.

28. The method according to claim 26 or 27, wherein the target DNA is detected by gel electrophoresis, blotting or sequencing, after amplification.

29. The method according to claim 2, wherein step 2(d) is carried out so that digestion does not occur within the known part of the target DNA sequence.

30. The method according to claim 29, wherein an oligonucleotide is only added to the digested end.