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Rolling circle amplification of DNA immobilized on solid surfaces and its application to multiplex mutation detection

1999; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês

10.1016/s1050-3862(98)00014-x

1879-1441

Anson V. Hatch, Takeshi Sano, John Misasi, Cassandra L. Smith,

Molecular Biology Techniques and Applications

A new method of amplifying short DNA molecules immobilized on a solid support has been developed. This method uses a solid-phase rolling circle replication reaction, termed rolling circle amplification (RCA). The probe consists of a single-stranded DNA primer anchored at the 5′ terminus to a solid support and a single stranded DNA template hybridized to the immobilized primer. Here, DNA ligase was used to circularize the template, and DNA polymerase I was used to extend the immobilized primer in a rolling circle replication reaction. This method was used to identify a known polymorphism in BRCA1 exon 5. These results demonstrate that RCA offers considerable promise to facilitate effective mutation screening of DNA using a solid-phase format.