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New Technology
Nature Genetics  19, 225 - 232 (1998)
doi:10.1038/898

Mutation detection and single-molecule counting using isothermal rolling-circle amplification

Paul M. Lizardi1, Xiaohua Huang2, Zhengrong Zhu2, Patricia Bray-Ward2, David C. Thomas3 & David C. Ward2

1  Department of Pathology, Yale University School of Medicine, 333 Cedar St. New Haven, Connecticut 06520, USA.

2  Department of Genetics, Yale University School of Medicine , 333 Cedar St. New Haven, Connecticut 06520, USA.

3  Oncor, Inc., 209 Perry Parkway, Gaithersburg, Maryland 20877, USA.

Correspondence should be addressed to Paul M. Lizardi paul.lizardi@yale.edu or David C. Ward ward@biomed.med.yale.edu
Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the − strand of DNA, a complex pattern of DNA strand displacement ensues that generates 109 or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

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Nature Genetics
ISSN: 1061-4036
EISSN: 1546-1718
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