Methods for detecting nucleic acid sequences

• US 7,468,245 B2
• Filed: 11/27/2002
• Issued: 12/23/2008
• Est. Priority Date: 06/24/1998
• Status: Expired due to Fees

• Current Assignee: Enzo Diagnostics, Inc.
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Donegan, James J., Rabbani, Elazar, Coleman, Jack, Walner, Marleen
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Tung; Joyce
• Application Number:
  US10/305,690
• Publication Number:
  US 20030165936A1
• Time in Patent Office: 2,218 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/23.1
• US Class Current: 435/6.15
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

This invention provides novel processes for amplifying nucleic acid sequences of interest, including linear and non-linear amplification. In linear amplification, a single initial primer or nucleic acid construct is utilized to carry out the amplification process. In non-linear amplification, a first initial primer or nucleic acid construct is employed with a subsequent initial primer or nucleic acid construct. In other non-linear amplification processes provided by this Invention, a first initial primer or nucleic acid construct is deployed with a second initial primer or nucleic acid construct to amplify the specific nucleic acid sequence of interest and its complement that are provided. A singular primer or a singular nucleic acid construct capable of non-linear amplification can also be used to carry out non-linear amplification in accordance with this invention. Post-termination labeling process for nucleic acid sequencing is also disclosed in this invention that is based upon the detection of tagged molecules that are covalently bound to chemically reactive groups provided for chain terminators. A process for producing nucleic acid sequences having decreased thermodynamic stability to complementary sequences is also provided and achieved by this invention. Unique nucleic acid polymers are also disclosed and provided in addition to other novel compositions, kits and the like.

Claims

1. A process for detecting the presence of a specific target nucleic acid sequence comprising the steps of:

1) providing one or more first initial primers or first initial nucleic acid constructs comprising at least two segments;

(A) a first segment being sufficiently complementary to a portion of said specific target nucleic acid sequence to (i) bind or hybridize thereto and (ii) provide for template dependent extension such that a first primer extension is produced and

(B) a second segment being (i) non-identical to said first segment, and (ii) sufficiently complementary to sequences that are synthesized by extension of the first segment of said first initial primers or first initial nucleic acid constructs with said specific target nucleic acid sequence as a template to bind or hybridize thereto;

wherein the portion of said specific target nucleic acid sequence that comprises sequences identical to the second segment of the first initial primer or initial nucleic acid construct and the portion of said specific target nucleic acid sequence that comprises sequences complementary to the first segment of said first initial primer or first initial nucleic acid construct are in close proximity to each other on said specific target nucleic acid sequence;

2) providing at least one second initial primer or second initial nucleic acid construct, wherein (i) said second initial primer or second initial nucleic acid construct comprises a first segment sufficiently complementary to said first primer extension to bind or hybridize thereto, (ii) wherein said second initial primer or second initial nucleic acid construct is nonidentical to said first initial primer or first initial nucleic acid construct (iii) wherein said first segment provides for template dependent primer extension such that a second primer extension is produced and (iv) wherein said second initial primer or second initial nucleic acid construction further comprises a second segment being (a) non-identical to the first segment of said second initial primer or said second initial primer construct and (b) sufficiently complementary to sequences primer or second initial nucleic acid construct with said first primer isothermal conditions;

3) mixing said first initial primer or first initial nucleic acid construct and said second initial primer or second initial nucleic acid construct with substrates, buffer, a template-dependent polymerizing enzyme and a sample to be tested for the presence of said specific target nucleic acid sequence;

4) incubating said mixture under isostatic or limited cycling conditions, extension of said first initial primers or first initial nucleic acid constructs;

5) forming at least one stem-loop structure by separating said first segment of said first initial primer or first initial nucleic acid construct from said specific target nucleic acid sequences by self-annealing between said second segment of said first initial primer or first initial nucleic acid construct and a segment formed after target template dependent extension of the first segment of said first initial primer or first initial nucleic acid construct and

6) detecting the presence of said stem-loop structures formed in said forming step (5), thereby detecting the presence of said specific target nucleic acid sequence or its complement.

2. The process of claim 1, wherein less than about 50 nucleotides separate the portion of said specific target nucleic acid sequence that is identical to the second segment of the first initial primer or first initial nucleic acid construct from the portion of said specific target nucleic acid that is complementary to the first segment of the first initial primer or first initial nucleic acid construct.

3. The process of claim 1, wherein less than about 10 nucleotides separate the portion of said specific target nucleic acid sequence that is identical to the second segment of the first initial primer or first initial nucleic acid construct from the portion of said specific target nucleic acid sequence that is complementary to the first segment of the first initial primer or first initial nucleic acid construct.

4. The process of claim 1, wherein the portion of said specific target nucleic acid that is identical to the second segment of the first initial primer or first initial nucleic acid construct is adjacent to the portion of said specific target nucleic acid sequence that is complementary to the first segment of the first initial primer or first initial nucleic acid construct.

5. The process of claim 1, wherein said template-dependent polymerizing enzyme is selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase and a combination of any of the foregoing.

6. The process of claim 1, wherein said first initial primers, first initial nucleic acid constructs, first primer extensions, second initial primers, second initial nucleic aid constructs or second primer extensions comprise one or more moieties selected from the group consisting of modified nucleotides, non-nucleic acid polymers, branched nucleic acids, inverted nucleic acids, peptide nucleic acids, spacer groups, a basic sites, detectable labels and a combination of any of the foregoing.

7. The process of claim 1, wherein said first or second initial primer or first or second initial nucleic acid construct or said first or second primer extension comprises one or more modified nucleotides having detectable labels.

8. The process of claim 7, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluorescent molecules, infra-red fluorescent groups, chemiluminescent moieties, enemy transfer systems, enzymes and a combination of any of the foregoing.

26. The process of claim 1 or 9, which further comprises providing additional primers.

27. The process according to claim 26, wherein said additional primers hybridize to a loop segment.

28. The process according to claim 26, wherein said additional primers are standard primers.

29. The process according to claim 26, wherein one or more of said additional primers comprises two segments, one segment thereof is complementary to sequences of said target nucleic acid and the other segment comprises sequences that are identical to said target nucleic acid.

30. The process according to claim 26, wherein extension of said additional primers displaces the first or second primer extension.

9. A process for detecting the presence of a specific target nucleic acid sequence comprising the steps of:

1) providing one or more first initial primers or first initial nucleic acid constructs comprising two segments;

(A) a first segment being sufficiently complementary to a portion of said specific target nucleic acid sequence to (i) bind or hybridize thereto and (ii) provide for template dependent extension such that a first primer extension is produced and

(B) a second segment being (i) non-identical to said first segment, and (ii) sufficiently complementary to sequences that are synthesized by extension of the first segment of said first initial primers or first nucleic acid constructs with said specific target nucleic acid sequences as a template to bind or hybridize thereto and wherein a stem-loop structure can be created by self-annealing between the second segment of the first initial primer or first nucleic acid construct and a segment formed after target template dependent extension of the first segment of the first initial primer or first nucleic acid construct wherein the stability of a stem formed by such intrastrand hybridization is substantially equal to or is higher than the stability of a hybrid formed between said loop segment and its complement by interstrand hybridization;

2) providing at least one second initial primer or second initial nucleic acid construct, wherein (i) said second initial primer or second initial nucleic acid construct comprises a first segment sufficiently complementary to said first primer extension to bind or hybridize thereto;

(ii) said second initial primer is nonidentical to said first primer or construct;

(iii) wherein said first segment provides for template dependent primer extension such that a second primer extension is produced and (iv) wherein said second initial primer or initial nucleic acid construct further comprises a second segment being (a) non-identical to the first segment of said second initial primer or second initial nucleic acid construct and (b) sufficiently complementary to sequences that are synthesized by extension of the first segment of said second initial primer or second initial nucleic acid construct with said first primer extension as a template to hybridize or bind thereto under isostatic or limited cycling conditions;

3) mixing said first initial primer or first initial nucleic acid construct and said second initial primer or second initial nucleic acid construct with substrates, buffer, a template-dependent polymerizing enzyme and a sample to be tested for the presence of said specific target nucleic acid sequence;

4) incubating said mixture under isostatic or limited cycling condition;

5) forming at least one stem-loop structure by separating said first segment of said first initial primer or first initial nucleic acid construct from said specific target nucleic acid sequence by self-annealing between said second segment of said first initial primer or first initial nucleic acid construct and a segment formed after target template dependent extension of the first segment of said first initial primer or first initial nucleic acid construct and

6) detecting the presence of said stem-loop structures formed in said forming step (5), thereby detecting the presence of said specific target nucleic acid sequence or its complement.

10. The process of claim 9 wherein less than about 50 nucleotides separate the portion of said specific target nucleic acid sequence that is complementary to the first segment of the first initial primer or first initial nucleic acid construct from the portion of said specific target nucleic acid sequence that is identical to the second segment of the first initial primer or first initial nucleic acid construct.

11. The process of claim 9, wherein less than about 10 nucleotides separate the portion of said specific target nucleic acid sequence that is complementary to the first segment of the first initial primer or first initial nucleic acid construct from the portion of said specific target nucleic acid sequence that is identical to the second segment of the first initial primer or first initial nucleic acid construct.

12. The process of claim 9, wherein the portion of said specific target nucleic acid sequence that is complementary to the first segment of the first initial primer or first initial nucleic acid construct is adjacent to the portion of said specific target nucleic acid sequence that is identical to the second segment of the first initial primer or first initial nucleic acid construct.

13. The process of claim 9, wherein less than about 50 nucleotides separate the portion of said first primer extension that is complementary to the first segment of the second initial primer or second initial nucleic acid construct from the portion of said first primer extension that is identical to the second segment of the second initial primer or second initial nucleic acid construct.

14. The process of claim 9, wherein less than about 10 nucleotides separate the portion of said first primer extension that is complementary to the first segment of the second initial primer or second initial nucleic acid construct from the portion of said first primer extension that is identical to the second segment of the second initial primer or second initial nucleic acid construct.

15. The process of claim 9, wherein the portion of said first primer extension that is complementary to the first segment of the second initial primer or second initial nucleic acid construct is adjacent to the portion of said first primer extension that is identical to the second segment of the second initial primer or second initial nucleic acid construct.

16. The process of claim 9, wherein said template-dependent polymerizing enzyme comprises a member selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase and a combination of any of the foregoing.

17. The process of claim 9, wherein said first or second initial primers, first or second nucleic acid constructs or first or second primer extensions comprise one or more moieties selected from the group consisting of modified nucleotides, non-nucleic acid polymers, branched nucleic acids, inverted nucleic acids, peptide nucleic acids, spacer groups, a basic sites, detectable labels and a combination of any of the foregoing.

18. The process of claim 9, wherein said first or second initial primer or first or second nucleic acid construct or first or second primer extension comprises one or more modified nucleotides having detectable labels.

19. The process of claim 18, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infrared fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

20. A process for detecting the presence of a specific target nucleic sequence comprising the steps of:

(a) amplifying said specific target nucleic sequence comprising the steps of;

1) providing

(i) said specific target nucleic acid sequence;

(ii) one or more first initial primers or first initial nucleic acid constructs comprising two segments;

(A) a first segment being sufficiently complementary to a portion of said specific target nucleic acid sequence (i) to bind or hybridize thereto and (ii) provide for template dependent extension such that a first primer extension is produced and

(B) a second segment being (i) non-identical to said first segment, and (ii) sufficiently complementary to sequences that are synthesized by extension of the first segment of said first initial primer or first initial nucleic acid construct with said specific target nucleic acid sequence as a template to bind or hybridize thereto;

wherein the portion of said specific target nucleic acid sequence that comprises sequences complementary to the first segment of the first initial primer or first initial nucleic acid construct and the portion of said specific target nucleic acid sequence that comprises sequences identical to the second segment of said first initial primer or first initial nucleic acid construct are in close proximity to each other on said target nucleic acid sequence;

(iii) one or more second initial primers or second initial nucleic acid constructs, wherein (A) said second initial primer or second initial nucleic acid construct comprises a first segment sufficiently complementary to said first primer extension to bind or hybridize thereto, (B) wherein said second initial primer or second initial nucleic acid construct is nonidentical to said first initial primer or first initial nucleic acid construct (C) wherein said first segment provides for template dependent primer extension such that a second primer extension is produced and (D) wherein said second initial primer or said second initial nucleic acid construct further comprises a second segment being (a) non-identical to the first segment of said second initial primer or said second initial primer construct and (b) sufficiently complementary to sequences that are synthesized by extension of the first segment of said second initial primer or second initial nucleic acid construct with said first primer extension as a template to hybridize or bind thereto;

(iv) substrates, buffer and a template-dependent polymerizing enzyme; and

2) incubating said specific target nucleic acid sequence and said first initial primer or first nucleic acid construct and said second initial primer or second initial nucleic acid construct under isostatic or limited cycling conditions in the presence of said substrates, buffer and template-dependent polymerizing enzyme, thereby producing an amplification product comprising two or more copies of said specific target nucleic acid sequence or its complement, wherein said second segment of said first initial primer or first nucleic acid construct and said specific target nucleic acid sequence complementary to said second segment are located at the same end of a nucleic acid strand of said two or more copies and

(b) detecting said amplification product.

21. The process according to claim 20, wherein said amplification product is detected by binding of a fluorescent intercalator.

22. The process according to claim 20, wherein said amplification product is detected by gel electrophoresis.

23. The process according to claim 20, wherein said amplification product is detected by a target specific nucleic acid probe.

24. The process according to claim 23, wherein said nucleic acid probe is labeled with a detectable label.

25. The process according to claim 24, wherein said detectable label is selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-red fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.