Modified nucleic acid polymers and methods for their production

• US 7,713,691 B2
• Filed: 11/27/2002
• Issued: 05/11/2010
• 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: Walner, Marleen, Stavrianopoulos, Jannis G., Donegan, James J., Rabbani, Elazar, Coleman, Jack
• Primary Examiner(s): Babic; Christopher M.
• Application Number:
  US10/305,664
• Publication Number:
  US 20030170682A1
• Time in Patent Office: 2,722 Days
• Field of Search: None
• 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

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 producing a single-stranded or double stranded modified nucleic acid polymer selected from the group consisting of a linear nucleic acid, branched nucleic acid, an inverted nucleic acid and a peptide-nucleic acid, or a combination of any of the foregoing, wherein said nucleic acid polymer comprises at least one modified purine or pyrimidine base, wherein said purine or pyrimidine base is modified with a negatively charged chemical moiety which comprises carboxylic acid covalently attached to an allyl moiety and wherein said polymer has decreased thermodynamic stability in binding to complementary sequences as compared to a corresponding unmodified nucleic acid polymer comprising the step of enzymatically incorporating into the nucleic acid sequences produced at least one modified nucleotide or nucleotide analog having a negatively charged chemical moiety, wherein said negatively charged chemical moiety comprises carboxylic acid covalently attached to an allyl moiety, wherein said nucleic acid is produced under thermocycling conditions where an annealing step and denaturation step are undertaken at temperatures that are no more than 12°

C. apart.

7. The method of claim 1, wherein said annealing and denaturation steps are no more than 8°

C. apart.

2. A process for producing a single-stranded or double stranded modified nucleic acid polymer selected from the group consisting of a linear nucleic acid, branched nucleic acid, an inverted nucleic acid and a peptide-nucleic acid, or a combination of any of the foregoing, wherein said nucleic acid polymer comprises at least one modified purine or pyrimidine base, wherein said purine or pyrimidine base is modified with a negatively charged chemical moiety which comprises carboxylic acid covalently attached to an allyl moiety and wherein said polymer has decreased thermodynamic stability in binding to complementary sequences as compared to a corresponding unmodified nucleic acid polymer comprising the step of enzymatically incorporating into the nucleic acid sequences produced at least one modified nucleotide or nucleotide analog having a negatively charged chemical moiety, wherein said negatively charged chemical moiety comprises carboxylic acid covalently attached to an allyl moiety wherein said nucleic acid is produced under thermocycling conditions where an extension step and denaturation step are undertaken at temperatures that are no more than 8°

C. apart.

8. The method of claim 2, wherein said annealing and denaturation steps are no more than 8°

C. apart.

3. A process for producing a single-stranded or double stranded modified nucleic acid polymer selected from the group consisting of a linear nucleic acid, branched nucleic acid, an inverted nucleic acid and a peptide-nucleic acid, or a combination of any of the foregoing, comprising producing a nucleic acid polymer under thermocycling conditions by means of a series of annealing and denaturation steps undertaken at temperatures that are no more than 12°

C. apart and enzymatically incorporating modified nucleotides or nucleotide analogs into said nucleic acid polymer, wherein said modified nucleotides or nucleotide analogs comprise a negatively charged chemical moiety, wherein said negatively charged chemical moiety comprises carboxylic acid and wherein said nucleic acid polymer produced has decreased thermodynamic stability in binding to complementary sequences as compared to a corresponding unmodified nucleic acid polymer due to the presence of said negatively charged chemical moiety in said modified nucleotides or nucleotide analogs incorporated into said nucleic acid polymer wherein the ability to produce said single-stranded or double-stranded nucleic acid polymer by a series of annealing and denaturation series that are no more than 12°

C. apart is derived from the presence of said carboxylic moiety.

5. The method of claim 3, wherein said annealing and denaturation steps are no more than 8°

C. apart.

4. A process for producing a single-stranded or double stranded modified nucleic acid polymer selected from the group consisting of a linear nucleic acid, branched nucleic acid, an inverted nucleic acid and a peptide-nucleic acid, or a combination of any of the foregoing, comprising producing a nucleic acid polymer under thermocycling conditions by means of a series of annealing and denaturation steps undertaken at temperatures that are no more than 12°

C. apart and enzymatically incorporating modified nucleotides or nucleotide analogs into said nucleic acid polymer, wherein modified nucleotides or nucleotide analogues are incorporated into said nucleic acid polymer, wherein said modified nucleotide or nucleotide analogs comprises a negatively charged chemical moiety, wherein said negatively charged chemical moiety comprises carboxylic acid and wherein said nucleic acid polymer produced has decreased thermodynamic stability in binding to complementary sequences as compared to a corresponding unmodified nucleic acid polymer due to the presence of said negatively charged chemical moiety in said modified nucleotides or nucleotide analogs incorporated into said nucleic acid polymer and wherein the production of said single-stranded or double-stranded modified nucleic acid polymer is detected by means of a fluorescent nucleic acid intercalator.

6. The method of claim 4, wherein said annealing and denaturation steps are no more than 8°

C. apart.