Ultrasensitive DNA detection based on target-triggered rolling circle amplification and fluorescent poly(thymine)-templated copper nanoparticles |
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Authors: | Kwan Woo Park Chang Yeol Lee Bhagwan S. Batule Ki Soo Park Hyun Gyu Park |
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Affiliation: | Department of Chemical and Biomolecular Engineering (BK 21+ Program), KAIST, Daehak-ro 291, Yuseong-gu, Daejeon 34141 Republic of Korea, Fax: +82-42-350-3910, +82-42-350-3932 ; Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029 Republic of Korea, Fax: +82-2-450-3742, +82-2-450-3742 |
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Abstract: | We describe a novel strategy for the ultrasensitive detection of target DNA based on rolling circle amplification (RCA) coupled with fluorescent poly(thymine)-templated copper nanoparticles (poly T-CuNPs). In the presence of target DNA, a padlock DNA probe that consists of two regions: a target DNA-specific region and a poly(adenine) region, is circularized by the ligation reaction, and the subsequent RCA reaction is promoted to generate long, concatemeric, single-stranded DNA (ssDNA) with a lot of repetitive poly T sequences. As a result, a large number of poly T-CuNPs are formed, exhibiting a highly fluorescent signal. However, in the absence of target DNA or in the presence of non-specific target DNA, the padlock DNA probe is not circularized and the subsequent RCA is not executed, leading to no production of fluorescent poly T-CuNPs. With this simple strategy, we successfully analyzed the target DNA with the ultralow detection limit of 7.79 aM, a value that is 3 or 7 orders of magnitude lower than those of previous RCA-based fluorescent DNA detection strategies. In addition, the developed system was demonstrated to selectively discriminate non-specific target DNAs with one-base mismatch, suggesting potential application in the accurate diagnosis of single nucleotide polymorphisms or mutations.An ultrasensitive DNA detection method is developed based on target-triggered rolling circle amplification coupled with fluorescent poly(thymine)-templated copper nanoparticles. |
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