Linear amplification of specific nucleic acid sequences

• US 6,743,605 B1
• Filed: 06/24/1998
• Issued: 06/01/2004
• Est. Priority Date: 06/24/1998
• Status: Expired due to Term

• 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): Whisenant, Ethan
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/104,067
• Time in Patent Office: 2,169 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/25.32, 536/24.3
• US Class Current: 435/91.2
• 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 linearly amplifying a specific nucleic acid sequence comprising the steps of:

1) providing;

said specific nucleic acid sequence,

initial primers or nucleic acid constructs comprising two segments,

(A) a first segment (i) being sufficiently complementary to a first portion of said specific nucleic acid sequence to bind or hybridize thereto and (ii) which provides for template-dependent extension, and

(B) a second segment being (i) substantially non-identical to said first segment, (ii) substantially identical to a second portion of said specific nucleic acid sequence, (iii) which binds to a complementary sequence of said second segment under isostatic or limited cycling conditions after a first complementary copy of said specific nucleic acid sequence has been produced by template dependent extension of said first segment, and (iv) thereby providing for subsequent binding of a first segment of another said initial primer or nucleic acid construct to said first portion of said specific nucleic acid sequence under isostatic or limited cycling conditons, such that after primer extension, a second complementary copy is produced and displaces said first complementary copy; and

substrates, buffer and a template-dependent polymerizing enzyme; and

2) incubating said specific nucleic acid sequence and said initial primers or nucleic acid constructs in the presence of said substrates, buffer and template-dependent polymerizing enzyme under isostatic or limited cycling conditions;

thereby linearly amplifying said specific nucleic acid sequence.

2. The process of claim 1, wherein said first segment, said second segment or said first primer extension comprises at least one modified nucleotide or nucleotide analog.

3. The process of claim 2, wherein said second segment comprises at least one modified nucleotide or nucleotide analog which increases the thermodynamic stability of said first segment to its complement in said primer extension.

4. The process of claim 2 or 3, wherein said modified nucleotide or nucleotide analog comprises an intercalating agent.

5. The process of claim 1, wherein said first segment or said first primer extension or both comprises at least one modified nucleotide or nucleotide analog.

6. The process of claim 5, wherein said modified nucleotide or nucleotide analog decreases the thermodynamic stability of said first segment or said primer extension to its complement.

7. The process of claim 6, wherein said modified nucleotide or nucleotide analog comprises a negatively charged chemical group.

8. The process of claim 7, wherein said negatively charged chemical group comprises carboxylic acid.

9. The process of claim 1, wherein said initial primer or nucleic acid construct comprises a linear nucleic acid, a branched nucleic acid, an inverted nucleic acid or a peptide-nucleic acid.