Analysis of a tri-allelic SNP.
Quantitative mutation analysis.
Quantitative mutation analysis in long sequence runs.
Two mutation types quantified in a single Pyrosequencing reaction.
Detection of tri- and tetra-allelic SNPs can be difficult with commonly used methods. This series of Pyrograms illustrates the ease of Pyrosequencing based detection of a tri-allelic SNP (red outline). C, T and G are serially dispensed in the Pyrosequencing reaction and only the incorporated nucleotides will elicit a signal peak. The result is a different peak pattern for homozygous samples of each allele (upper three Pyrograms) or compound peak patterns for heterozygous samples (lower three Pyrograms).
Pyrogram peak heights are proportional to the frequency of an allele in the sample. Therefore, it provides accurate measures of the proportion of, for example, a mutation in a blood sample.
Since single nucleotide polymorphisms (SNPs) are often not close to one another, common Pyrosequencing chemistry usually requires separate assays for each mutation site to be analyzed. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of more than one SNP in the same run. This example shows the analysis of a 10:90 mixture of wild-type and mutated EGFR. Even after 60 dispensations, the SNP analysis is exact.
Pyrogram of a DNA sequence featuring an insertion-deletion mutation (ATCTGCCC) and a somatic mutation involving a single base pair substitution (C vs. T). The variable regions are highlighted in blue and the allele frequencies are given above the indicated sites. The histogram (lower graph) indicates the number of nucleotides incorporated at each nucleotide dispensation. The dark blue bars represent the nucleotide positions conserved between alleles and arrowed empty bars portray the quantified variation.