In vitro amplification of nucleic acid molecules via circular replicons

• US 20040191816A1
• Filed: 01/16/2004
• Published: 09/30/2004
• Est. Priority Date: 08/04/1992
• Status: Abandoned Application

• Current Assignee: Replicon Incorporated (Roper Technologies, Inc.)
• Original Assignee: Replicon Incorporated (Roper Technologies, Inc.)
• Inventors: Auerbach, Jeffrey I.
• Application Number:
  US10/759,644
• Publication Number:
  US 20040191816A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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 2521/501 Ligase
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

Methods and compositions suitable for accomplishing the in vitro amplification of nucleic acid molecules via enzymatic means are provided. The preferred means employ circular rather than linear replicons. Means for producing such circular replicons from linear reactants are also provided.

Claims

1-20. -20 (canceled).

21. A composition for amplifying a target polynucleotide region of a nucleic acid molecule, the composition comprising:

(a) a sample comprising a component, or extract thereof, selected from the group consisting of blood, stool, sputum, mucus, serum, urine, saliva, tear, biopsy material, tissue, PAP smear, mole, wart, agricultural product, waste water, drinking water, milk, processed food, and air;

(b) a single-stranded first polynucleotide comprising a polynucleotide region that is complementary in sequence to a target polynucleotide region of a nucleic acid molecule of said sample, wherein said first polynucleotide is circular or is circularizable when hybridized to a polynucleotide comprising said target polynucleotide region;

(c) a second polynucleotide comprising a 3′

terminus hybridized to said single-stranded first polynucleotide and comprising said target polynucleotide region;

(d) a third polynucleotide comprising a polynucleotide region comprising a sequence that is complementary to a region of said second polynucleotide, and whose 3′

terminus is hybridized thereto; and

(e) a polymerase that extends the 3′

termini of said second and third polynucleotides in a template-dependent manner, thereby to provide exponential amplification of said target polynucleotide region.

22. A transgenic animal comprising a circularized nucleic acid molecule produced through the in vitro amplification of an in vitro composition, the composition comprising:

(a) a single-stranded first polynucleotide comprising a polynucleotide region comprising a nucleic acid sequence that is complementary to a target polynucleotide region of a nucleic acid molecule of a human or non-human mammalian gene, and is circular or is circularizable when hybridized to a polynucleotide comprising said target polynucleotide region;

(b) a second polynucleotide comprising a 3′

terminus hybridized to said single-stranded first polynucleotide and comprising said target polynucleotide region;

(c) a third polynucleotide comprising a polynucleotide region that is complementary in sequence to a region of said second polynucleotide, and whose 3′

terminus is hybridized thereto; and

(d) a polymerase that extends the 3′

termini of said second and third polynucleotides in a template-dependent manner, thereby to provide exponential amplification of said target polynucleotide region.