Mutation Analysis and Genotyping

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Pyrosequencing for quantitative analysis of sequence variations
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Improve the detection range of your assays, boost the discriminatory power of your analyses, and expand the spectrum of genetic variation that you can examine. Pyrosequencing can enhance genetic analyses by delivering explicit and quantitative sequence data. Furthermore, independent studies have demonstrated that Pyrosequencing shows full concordance with Sanger sequencing, but is more cost-effective and less labor-intensive.
Strengths of Pyrosequencing
Pyrosequencing for quantitative sequence variation analysis
Analysis of complex mutations
Strengths of Pyrosequencing
Pyrosequencing is accurate, sensitive, and flexible. It enables:
  • Reliable detection and quantification of sequence variation, even when mutations are found in low proportions
  • Characterization of contiguous and multivariable mutations
  • Discovery of unknown mutations
  • Rapid allele quantification in loss of heterozygosity (LOH) analyses
  • Unambiguous, fully quantitative genotyping that distinguishes multi-site variations from single nucleotide polymorphisms

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Pyrosequencing for quantitative sequence variation analysis
Genetic testing is an important component of many applications. For example, developing effective therapeutic agents requires information about how gene polymorphisms impact metabolism; understanding genetic contributions to a disease involves characterizing linked mutations; finally, analysis of forensic DNA evidence relies on accurate detection of sequence variation.

Many mutation types are important in fields such as epidemiology, pharmacogenetics, and animal husbandry, and new markers are continuously being discovered. As a result, a variety of analysis methods are used to detect genetic variability. This can complicate comparison of results obtained by different methods and different researchers. Ideally, a single technology could be adapted for all applications to standardize results. Pyrosequencing offers precisely that versatility. Because of the flexibility of primer placement in Pyrosequencing reactions, virtually all genetic markers — those currently in use as well as those yet to be identified — can be assayed. Alleles of variable loci are accurately quantified, and heterozygosity is easily resolved (see figures Analysis of a tri-allelic SNP and Quantitative mutation analysis). In addition, because Pyrosequencing delivers sequence information, various types of genetic variation can be evaluated — insertion-deletions, single nucleotide polymorphisms, single tandem repeats, and variable gene copy number — and it is possible to assay several contiguous sequence variants in a single run.

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Analysis of complex mutations
Regardless of the marker or mutation being analyzed, preparation of templates for Pyrosequencing and the subsequent analysis of the resulting sequence information are quick and easy, saving time and valuable resources. However, the strength of Pyrosequencing for genetic testing lies in the elegance of its output. Because results are simply the true sequence of the DNA in a sample, the user can examine multiple mutation sites within a specified region, and even multiple variation types, all in the same run (see figure Two mutation types quantified in a single Pyrosequencing reaction). The improved chemistry of the PyroMark Q24 Advanced system also enables analysis of multiple mutations over long sequences (see figure Quantitative mutation analysis in long sequence runs).

Furthermore, the straightforward results are easily interpreted. Pyrosequencing enables de novo sequencing which, coupled with the built-in control afforded by the sequence surrounding the variable site, is a guaranteed way of validating newly identified markers. The high throughput facilitates rapid compilation of the population data needed to establish reference databases for these markers. This feature makes Pyrosequencing a powerful and versatile tool for the development of pharmacogenetic markers (1).
  1. Koontz, D.A., Huckins, J.J., Spencer, A., and Gallagher, M.L. (2009) Rapid detection of the CYP2A6*12 hybrid allele by Pyrosequencing technology. BMC Med. Gen. 10, 80.

Analysis of a tri-allelic SNP
Analysis of a tri-allelic SNP.
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).
Quantitative mutation analysis
Quantitative mutation analysis.
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.
Quantitative mutation analysis in long sequence runs
Quantitative mutation analysis in long sequence runs.
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.
Two mutation types quantified in a single Pyrosequencing reaction
Two mutation types quantified in a single Pyrosequencing reaction.
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.