Mutation Detection and Genotyping
Mutations in PCR products are detected by the presence of heteroduplexes optimally detected by our LCGreen® Plus dye. Variations are identified by changes in the shape of the melting profile compared to a reference sample.
Hi-Res Melting® on the LightScanner® and LightScanner 32 Systems are being used for mutation discovery in tumor samples, yielding information on mutations present in low allele fractions (as low as 5%) and allelic imbalances between tumor and matched normal samples.
Genotyping is possible with Hi-Res Melting eliminating the need for labeled probes. It is achieved by Hi-Res Melting® in two ways:
- With unlabeled 3' blocked probes data from the probe and PCR product can be obtained simultaneously.
- Small amplicons often as short as 50 ~ 60 bp with primers just flanking the SNP. As this application relies on subtle differences in melting temperature, it is best performed on our instruments which have the best temperature precision on the market for melting.
The LightScanner is the first and only instrument to utilize Hi-Res Melting® for mutation scanning and genotyping in a 96- or 384-well plate system. This exciting technology, licensed exclusively to BioFire Diagnostics, is based on U.S. Patent No. 7,387,887 (Nucleic Acid Melting Analysis with Saturation Dyes) and U.S. Patent Publication 2006-0019253 (Amplicon Melting Analysis with Saturation dyes).
The LightScanner is unsurpassed in its temperature control and data acquisition rates. With superb sensitivity and specificity, this instrument is designed to meet the needs of high throughput scanning projects. Simply add LCGreen® dye to the initial PCR reaction– then transfer the PCR plate to the LightScanner for melting. Intuitive analysis software groups the results and provides automatic results.
LightScanner® 32 System
The LightScanner 32 (LS32™) instrument is an air thermocycler capable of automatically analyzing samples for the presence of targeted nucleic acid. It combines real-time, rapid thermocycling technology and Hi-Res Melting and is capable of analyzing 32 samples per run in less than 60 min.
The LS32 instrument performs three-color detection for real-time and single-color detection for Hi-Res Melting. Real-Time PCR results can be generated using a variety of labeled probe chemistry including SimpleProbe®, FRET probes or HybProbe®. Optimal Hi-Res Melting results are generated using LCGreen® Plus dye.
Like the LightScanner, the LS32 is unsurpassed in its temperature control and data acquisition rates. With superb sensitivity and specificity, this instrument is designed to meet the needs of high throughput scanning projects. Intuitive analysis software groups the results and provides automatic results.
LCGreen® Plus+ Melting Dye
LCGreen dye is specifically designed for Hi-Res Melting curve analysis to detect DNA sequence variants. The addition of LCGreen dye increase the melting temperature of DNA by 1-3 °C and may require adjustment of cycling parameters. This dye is manufactured exclusively by BioFire Diagnostics and its chemical structures are unique among the scientific and patent literature.
LCGreen dye is tailored specifically for Hi-Res Melting and has the unique ability to detect heteroduplexes during melting analysis after PCR. Just add the dye to your sample before PCR. LCGreen dye is extremely stable, does not inhibit PCR, and is a "saturation" dye which makes it possible to detect multiple PCR products in a mixture during melting analysis.