American Home Products Corporation

Manoj Samuel

C Satishchandran

Catherine Julia Pachuk

Chain reaction cloning methods and reagents and kits for performing such methods are provided. Chain reaction cloning allows ligation of double-stranded DNA molecules by DNA ligases and bridging oligonucleotides. Double-stranded nucleic acid molecules are denatured into single-stranded molecules. The ends of the molecules are brought together by hybridization to a template. The template ensures that the two single-stranded nucleic acid molecules are aligned correctly. DNA ligase joins the two nucleic acid molecules into a single, larger, composite nucleic acid molecule. The nucleic acid molecules are subsequently denatured so that the composite molecule formed by the ligated nucleic acid molecules and the template cease to hybridize to each. Each composite molecule then serves as a template for orienting unligated, single-stranded nucleic acid molecules. After several cycles, composite nucleic acid molecules are generated from smaller nucleic acid molecules. A number of applications are disclosed for chain reaction cloning including site-specific ligation of DNA fragments generated by restriction enzyme digestion, DNAse digestion, chemical cleavage, enzymatic or chemical synthesis, and PCR amplification.

Chain reaction cloning using a bridging oligonucleotide and DNA ligase

Methods of detecting the presence of linear DNA in a sample that contains circular DNA are disclosed. The methods comprise combining an ATP-dependent deoxyribonuclease, ATP and a sample to form a reaction mixture, maintaining the reaction mixture under conditions in which linear DNA is processed by the ATP-dependent deoxyribonuclease, and detecting the presence of ADP thus produced. Methods of detecting the presence of DNA, RNA, or protein in samples are disclosed. The methods comprise combining an ATP-dependent deoxyribonuclease, an ATP-dependent ribonuclease, or an ATP-dependent protease, respectively, with ATP and a sample to form a reaction mixture. The reaction mixture is maintained under conditions in which DNA, RNA or protein is processed by the respective ATP-dependent enzyme. The presence of ADP thus produced is detected, indicating the presence of the enzyme substrate. Methods of quantifying the DNA molecules, RNA molecules or protein molecules are also disclosed.

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West Chester, PA, United States of America

Manoj Samuel