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The superRCA® assay begins with sampling genomic DNA (gDNA) or circulating tumor DNA (ctDNA) from blood or bone marrow aspiration, followed by target gene amplification through limited Polymerase Chain Reaction (PCR) cycles. This process amplifies the DNA sequences of interest that are known to be mutated in malignant cells. |
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The process starts with the denaturation of the DNA amplicon to produce single-stranded DNA (ssDNA) fragments. This is followed by the addition of a specifically designed oligonucleotide, serving as a ligation template, with 3’ and 5’ ends complementary to the corresponding 3’ and 5’ ends of the ssDNA fragments. This enables the 3’ and 5’ ends of ssDNA to come into close proximity with each other. |
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A ligase enzyme then catalyzes the ligation of these ends, forming a circular DNA molecule containing the target sequence. As the process continues, the oligonucleotide not only acts as the ligation template but also serves as a primer for the initial RCA process. RCA is an isothermal DNA amplification process that uses a circular DNA template, a strand-displacing DNA polymerase, and a primer to generate a long, single-stranded DNA product. The product contains multiple tandem repeats, each complementary to the circular DNA template. |
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These RCA products are subsequently interrogated using another set of genotyping probes: one for the wild-type allele and one for the mutant allele. These probes, called padlock probes, are short DNA oligonucleotides with segments at the 3’ and 5’ ends that are complementary to a target region. Upon hybridization, the two ends of the probe are oriented in juxtaposition on the target template, leaving a nick site. DNA ligase catalyzes nick closure, circularizing the probe only when its termini exhibit perfect complementarity to the target sequence, enhancing the single-base discrimination capacity of the padlock probes. |
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The resulting circular probe serves as a template for superRCA® amplification. The simultaneous use of wild-type and mutant probes in a single reaction enables accurate quantification of allelic variants. For each starting DNA amplicon, the reaction gives rise to large clusters of single-stranded DNA superstructures called superRCA® products. |
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The superRCA® products can be scored as mutant- or wild-type-specific using fluorophore-labeled hybridization probes. Up to a million hybridization probes can bind to each mutant- or wild-type-specific superRCA® product, allowing for efficient visualization and analysis via standard flow cytometry. |
