2012 © Institute for Molecular Medicine Finland FIMM
Roche LightCycler 480 SNP Genotyping
LightCycler SNP genotyping is a Real-Time Polymerase Chain Reaction (RT PCR) where amplification of targeted DNA fragment can be followed as it occurs (meaning in real time). This enables rapid SNP genotyping in only a few hours with no further applications needed. This method is applied for custom genotyping of interesting SNPs and samples can be placed in 96 or 384 well plate format. The main application of this method is to genotype a few SNPs in large number of samples (several hundreds to thousands).
LightCycler 480 system has two different methods of SNP analysis available:
-
Endpoint genotyping analysis
Endpoint genotyping analysis uses two sequence-specific probes (e.g. TaqMan probes) that are designed for wildtype and mutant target DNA and these probes are labeled with different fluorescent dyes. Each probe contains two labels (a fluorescent reporter and a quencher) which are in close proximity to each other. When the probe is intact, the quencher can suppress the reporter signal. During the elongation process the probe is cleaved thus separating the reporter and the quencher from each other and that is seen as a fluorescent signal from the reporter. The software determines the genotype of the sample by measuring the intensity distribution of used dyes after PCR.
-
Melting Curve genotyping analysis
Melting curve analysis looks at the temperature at which the DNA double helix strands separate from each other or melt. This temperature is dependent on the content and length of the DNA sequence. In the melting curve genotyping experiment labeled sequence-specific probes are added together with PCR primers to amplification process and they are allowed to anneal to the reaction products. The melting curve is acquired by heating the probe-amplicon heteroduplex and measuring the changes in fluorescence that results when probe melts from the amplicon. Even a single mismatch (e.g. a SNP) between probe and amplicon will show a reduced melting temperature compared to the perfect match.
Our service includes all steps from the assay/SNP order through to genotyping and quality control.