Integration of rolling circle amplification and cationic conjugated polymer for the homogeneous detection of single nucleotide polymorphisms

Chinese Science Bulletin (Impact Factor: 1.58). 11/2011; 56(31):3247-3252. DOI: 10.1007/s11434-011-4663-0


A novel, homogeneous and sensitive assay for the detection of single nucleotide polymorphisms (SNPs) by integration of rolling
circle amplification (RCA) and cationic conjugated polymer (CCP) has been developed and tested. Mutant DNA serves as the template
for specifically circularizing a padlock probe (PLP) with a sequence that is complementary to the mutant DNA. Afterwards,
the mutant DNA directly acts as the primer to initiate the RCA reaction in the presence of phi29 DNA polymerase that generates
a long, tandem single-strand DNA product. During the RCA reaction, fluorescein-labeled dUTPs are incorporated into the RCA
products. When the CCP is introduced, efficient FRET from CCP to fluorescein occurs as a result of the strong electrostatic
interactions between the CCP and the DNA produced by RCA. The wild-type DNA contains a single base mismatch with PLP with
the result that the PLP is not circularized, RCA is not triggered and inefficient FRET results. By measuring the change of
the emission intensities of CCP and fluorescein, it was possible to detect the SNP in a homogeneous manner. The method is
sensitive and specific enough to detect 0.1 pmol/L mutant DNA and to determine a mutant allele frequency as low as 2.0%.

Keywordsrolling circle amplification–cationic conjugated polymer–single nucleotide polymorphisms–homogeneous detection

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