Signature Technology
The Signature® technology is optimized for the rapid multiplex analysis of nucleic acid sequences. Up to 100 DNA or RNA targets can be detected in a single reaction. With medium density capability and a streamlined workflow, the Signature products allow for rapid detection of multiple analytes in a single assay, increase the number of assays available on a single platform, and reduce the overall turn around time
Following extraction of nucleic acids from the specimen of interest, the Signature procedure consists of three simple steps (Figure 1):
- Multiplex PCR amplification (or RT-PCR in the case of RNA-based assays)
- Hybridization of PCR products onto specific capture probes
- Multiplex detection of probe-bound PCR products
These simple steps require minimal hands-on time, with results available in less than 6 hours. A typical workflow is shown in the schematic below.
Figure 1: Example of Signature Workflow for Rapid Multiplex RT-PCR Amplification and Detection
For RNA-based assays, the first step in the procedure is to perform a multiplex reverse transcription for all targets of interest. The resulting cDNAs are then amplified in a single multiplex PCR reaction using PCR primers specific for each individual cDNA. The resulting PCR products, or amplicons, are then hybridized to sequence specific oligonucleotides immobilized onto beads, and analyzed on the Luminex® 100ä IS or 200ä System. This procedure is illustrated in Figure 2.
Figure 2: Illustration of the major steps in a typical multiplex RNA amplification and detection reaction. In this example, a fusion transcript RNA is amplified by sequence-specific PCR primers. One of the primers is 5’ modified with a biotin. Following hybridization on a capture probe specific for the fusion transcript sequence, the PCR product is detected by addition of labeled streptavidin (SAPE).
For the detection step, Signature products utilize medium-density liquid bead array chemistry to provide a very sensitive method for multiplex detection of RNA or DNA targets. Each reaction is a bioassay on the surface of a color-coded bead that is uniquely identified by a red:infrared dye ratio, for a total of 100 different beads available per reaction (Foigure 3). Each of these color-coded beads can be covered with a covalently attached sequence-specific capture probe. When a nucleic acid sequence or gene is amplified by PCR utilizing biotin-labeled primers or dNTPs, a biotinylated PCR product is created. This PCR product can then hybridize to the color-coded bead via the sequence-specific capture probes.
Figure 3: Principle of Multiplex Bead-Based Hybridization for Signature Assays. The PCR products bound on each unique bead are subsequently labeled by streptavidin-phycoerythrin (SAPE) and detected by two lasers as the beads pass through the flow cell of the Luminex platform (Figure 4). The red laser identifies the color-coded bead, which specifies the capture probe. The green laser then detects the median fluorescence intensity (MFI) generated by the bound SAPE and reports whether a given PCR product is bound to a specific probe. By multiplexing the PCR reaction and the number of unique beads used, this format allows for detection of up to 100 individual analytes in a single well in less than 5 hours.
Figure 4: Principle of Multiplex Bead-Based Detection for Signature Assays.
In conclusion, Signature technology provides comprehensive information in a single well, reducing the need for and cost of maintaining numerous platforms and assays. Signature assays have simple workflows, and require minimal hands on time, with an average time to result of less than 6 hours. Signature products utilizing this technology include tests for genetic diseases, such as cystic fibrosis, as well as leukemia such as the Signature LTx (Leukemia Translocation Panel) (RUO)*. For more information on Signature products and technology, contact Asuragen at www.asuragen.com.
* For Research Use Only. Not for use in diagnostic procedures.
