Roche 480 SystemFrequently Asked Questions (FAQ) |
|
How does the Roche 480 work?
The LightCycler® 480 instrument makes use of fluorescent dyes for real-time monitoring of both the generation of PCR product during cycling and melting of PCR products in post-PCR melting curve analysis. Fluorescent signals measured during cycling are correlated with the amount of PCR product in a reaction (possible with both sequence independent and sequence-specific detection formats). Fluorescent measurements taken post-PCR during melting curve analysis are used for product characterization (sequence independent and sequence-specific detection formats), or genotyping (sequence-specific detection formats). The LightCycler® 480 supports several fluorescent analysis formats and can use a broad range of probes and dyes. (2007, Roche Diagnostics) How can the Roche 480 used for SNP detection? Utilizing sequence-specific probes, fourophores coupled to sequence-specific oligonuclelotide probes hybridize to their complementary sequence in target PCR products. Single-labeled probes use Roche SimpleProbe chemistry. Hybridization probes use Roche HybProbe chemistry. Hydrolysis probes use 5’-nuclease assays. (2007, Roche Diagnostics) How can the Roche 480 used for SNP discovery? “Gene scanning” or “mutation scanning” techniques detect the presence of sequence variation in target-gene derived PCR amplicons. "Gene Scanning" is based on "High Resolution Melting", a novel, closed-tube post-PCR method enabling genomic researchers to analyze genetic variations in PCR amplicons prior to, or as an alternative to sequencing. High Resolution Melting provides high specificity, sensitivity and convenience at significantly higher speed and much lower cost than other established (e.g., gel-based) methods. (2007, Roche Diagnostics) Sample DNA is first amplified by real-time PCR in the presence of LightCycler® 480 High Resolution Melting Dye. Immediately after DNA amplification, a High Resolution Melting cycle can be performed on the same instrument and analyzed with LightCycler® 480 Gene Scanning Software to identify sequence variants. The entire experiment can be done on the LightCycler® 480 Instrument; post-PCR analysis does not require a separate device. (2007, Roche Diagnostics) How will I learn to use the Roche 480? The IGMC will provide user training on the instrument and software. How much will it cost to use the Roche 480? Through a generous grant from the USDA, IGMC will be able to provide the equipment and services to Center users at an affordable rate. The rate to use the instrument only is $20.00/hour. Considering a plate holds 384 wells and a run typically lasts an hour the cost of run/reaction is $0.05. What supplies/services will the IGMC provide? The Center will perform all system maintenance and provide users with technical and troubleshooting advice. The Center will also maintain all user data on our server for backup and store large data files. The Center can also supply consumables for assays at cost; however users have no obligation to purchase them from the Center. Does the IGMC have protocols for SNP detection using the Roche 480? We have several established protocols, we can incorporate to design specific individual protocols depending on the type of information you want to generate. The Center manager will be able to guide users with protocol design. The Center also has reference articles for protocol development available. What size and type microplate can I use? The standard plate for the 480 is a Roche product, #04-729-749-001 and sealing film #04-729-757-001 both are available through the Center. How long will it take to run an assay? The actual microplate assay will depend on PCR program, most take between 1 and 2 hours. Preparing samples for assays can easily be done in the Center. We have equipment which is available for Center users that aids in assembling reactions, including Matrix multichannel repeater dispensers and a Hydra® Microdispenser. How can I get started? Contact IGMC for more information.        |