Method for detecting nucleic acid sequences

• US 8,486,633 B2
• Filed: 10/26/2007
• Issued: 07/16/2013
• Est. Priority Date: 06/24/1998
• Status: Expired due to Term

• Current Assignee: Enzo Diagnostics, Inc.
• Original Assignee: Enzo Diagnostics, Inc.
• Inventors: Rabbani, Elazar, Stavrianopoulos, Jannis G., Donegan, James J., Coleman, Jack, Walner, Marleen
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US11/925,730
• Publication Number:
  US 20090263789A1
• Time in Patent Office: 2,090 Days
• Field of Search: 435/91.1, 435/91.2, 435/6, 435/6.12
• US Class Current: 435/6.12
• 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

The invention is directed to a method for detecting a target nucleic acid sequence by non-linear amplification.

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

(B) a second segment being (i) substantially non-identical to said first segment, (ii) substantially identical to a portion of said specific target nucleic acid sequence, (iii) 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 and 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 substantially complementary to the second segment of said first initial primer or first initial nucleic acid construct are in close proximity to each other;

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 or second initial nucleic acid construct is nonidentical to said second segment of said first initial primer or first initial nucleic acid construct and (iii) said first segment provides for template dependent primer extension such that a second primer extension is produced;

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, such that, if said target nucleic acid sequence is present in said sample, extension of said first initial primers or first initial nucleic acid constructs occurs;

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

(B) a second segment being (i) substantially non-identical to said first segment, (ii) substantially identical to a portion of said specific target nucleic acid sequence, (iii) 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 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 a loop segment in said stem-loop structure 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 or second initial nucleic acid construct is nonidentical to said second segment of said first initial primer or first initial nucleic acid construct and (iii) wherein said first segment provides for template dependent primer extension such that a second primer extension is produced;

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;

5) if said target nucleic acid sequence is present in said sample, 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.

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

(B) a second segment being (i) substantially non-identical to said first segment, (ii) substantially identical to a portion of said specific target nucleic acid sequence, (iii) 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 and 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 substantially identical to the second segment of said first initial primer or first initial nucleic acid construct are in close proximity to each other;

(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) said second initial primer or second initial nucleic acid construct is nonidentical to said second segment of said first initial primer or first initial nucleic acid construct and (C) said first segment provides for template dependent primer extension such that a second primer extension is produced;

(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.