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Genotyping (SNP analysis) on the LightCycler® 480 System can be done based on two different methods:
For both methods, software modules for fully automated analysis are included with the LightCycler® 480 Software 1.5.
A third method, High Resolution Melting (HRM) analysis, is also available on the LightCycler® 480 System after installation of a separate software module, the LightCycler® 480 Gene Scanning Software. Although its use for genotyping has been widely documented in the literature, it is recommended for mutation discovery rather than detection/genotyping purposes.
With Melting Curve Analysis, different alleles or allele combinations are identified due to the differing interaction strengths they have with the probe. Allele-specific primers or probes are not needed; the same sequence is used for all alleles of an investigated SNP, or can even cover several nearby SNPs. This saves reagent costs and, combined with the LightCycler® 480 Instrument´s high number of available detection channels, enables straightforward reaction multiplexing. As a post-PCR process, melting curve analysis depends neither on the efficiency of the amplification process nor on the cleavage of a substrate (e.g., a dye attached to a probe) and is therefore, very robust. The LightCycler® 480 Genotyping algorithm works by grouping samples with similar melting curve shape either by auto-calling or via included standards of known genotype, and calls genotypes of unknown samples instantly based on their melting profile´s comparison to the identified groups.
Figure 1: The LightCycler® 480 Software Genotyping module allows automated grouping of related sequences (e.g., wild-type, homo- and heterozygote mutants) based on melting curve shape. Matching colors for samples in plate view and chart peaks allow easy visualization of results.
Figure 2: The capability of the LightCycler® 480 System to differentiate class 4 SNPs (A to T exchange) was investigated. Primers were designed to generate a 145 bp amplicon covering a SNP in the TNF alpha gene (rs723858, frequency A/T 70/30). 20 samples of total human genomic DNA were screened. Three different groups comprising homozygote (A or T; blue and green) and heterozygote (A and T; red) variants could be easily identified and differentiated based on melting curve shape. The LightCycler® 480 High Resolution Melting Master Mix and the LightCycler® 480 Gene Scanning Software were used in this experiment.
Figure 3: A polymorphism in the ADIPOR1 gene was analyzed with SimpleProbe probes. Melting curves and 3 genotypes (homozygous C/C and T/T, heterozygous C/T) are shown.
Figure 4: 96 different samples of total human genomic DNA (10 ng/µl) were applied to four adjacent wells of a 384 multiwell plate (e.g., sample #1 to wells A1, A2, B1, B2). Primers and SimpleProbe probes allowing the detection of four different SNPs were added to the wells in a checkerboard pattern, allowing the genotyping of all SNPs in all samples simultaneously. For all studied SNPs, both homozygous as well as heterozygous samples were found. Results are shown based on the use of the checkerboard subset feature included in the analysis software. MDR: multidrug resistance protein; LPLH: lipoprotein lipase H; ADD:adenosine deaminase; ADR: adrenergic receptor
Figure 5: Melting curves for different alleles and allele combinations, obtained with the depicted, internally labeled SimpleProbe probe.
Figure 6: Different allelic combinations of the investigated SNPs, illustrated by individual melting curves of samples with known genotype.
Figure 7: Allele calling result in plate view, obtained using the genotyping module of the LightCycler® software in “high sensitivity” mode.
Figure 8: Allele calling result in table view. Score and resolution values indicate the accuracy and confidence of the automated calls.
Figure 9: 96 different samples of total human genomic DNA (10 ng/µl) were subjected to amplification and subsequent melting curve analysis, with primers and HybProbe probes allowing the detection of two adjacent SNPs in the Apolipoprotein B gene at 640 nm. Samples having the same allele combination (haplotypes) are shown on the right (note that double mutants with sequence variant TAG were not present).
Endpoint genotyping is based on the use of dual color hydrolysis probes (e.g., with commercially available predefined SNP genotyping assays). Genotypes can be called automatically and easily visualized in scatter plots. Amplification curves for both used channels can be displayed alternatively.
Figure 10: Example of SNP analysis using the Endpoint Analysis module of LightCycler® 480 Software 1.5. Genotype information is derived from the whole amplification process rather than from a post-PCR melting step.
Figure 11: Automated Scatter Plot Analysis.
Data source: all from Roche data on file
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