Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA |
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Authors: | Kyung-Tae Kim Chi-Bom Chae |
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Institution: | Department of Biomedical Science and Technology, Institute of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Korea. |
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Abstract: | In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization
with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or
glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple
strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated
by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product
was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared
to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive
to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes
immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA
was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis.
Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes
immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA.
Such a system should be advantageously applied for development of automated process for detection of DNA. |
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Keywords: | asymmetric PCR integrated PCR and hybridization self-assembly of branched DNA symmetric PCR |
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