Digital PCR: Rare Allele Applications with QuantStudio™ 3D Digital PCR System скачать в хорошем качестве

Digital PCR: Rare Allele Applications with QuantStudio™ 3D Digital PCR System

Learn more at http://www.lifetechnologies.com/quant... Genetic mutation is a hallmark of cancer, and the ability to detect genomic DNA changes is paramount to understanding this disease at a molecular level. In many cases, genetic changes can be as small as a single base substitution, or SNP, and this rarity poses an additional challenge for researchers. Assays developed to detect oncogenic mutations need to be able to identify just a few mutant cells in a huge abundance of wild type cells. This "finding a needle in a haystack" requires an assay that delivers both high signal-to-noise and low false positive/false negative rates. Common SNP genotyping technologies, such as CE sequencing and real-time PCR, are most effective in detecting mutant cells at prevalence no lower than about 20% (or approximately 1 in 5 cells). But, by combining available real-time PCR chemistries, such as TaqMan® Assays, with digital PCR, researchers are able to detect mutant cell prevalence down to 1%—and below So how does this work? First, let's review the basics of a TaqMan SNP Genotyping Assay. These assays are designed to resolve inherited SNP markers for which 3 genetic states are possible -- homozygous for allele 1, homozygous for allele 2, or heterozygous (containing one copy of each allele). The assay utilizes two gene specific primers and two allele-specific probes labeled with VIC and FAM dyes respectively. In the example shown, the presence of allele 1 is detected by VIC signal emission due to cleavage of the allele 1 probe. Similarly, the presence of allele 2 is detected by FAM signal emission due to cleavage of the allele 2 probe. The three possible outcomes are best viewed using a two-dimensional cluster plot. Because inherited germ line SNPs are either 0%, 50%, or 100% in normal diploid samples, the assays do not require extremely high probe hybridization specificity for assigning genotypes to individuals. So how can we use an inherently low-specificity assay to detect these rare events? The answer is to subdivide our sample in a digital PCR approach. Prior to PCR cycling, digital PCR requires partitioning of the DNA sample into hundreds or even thousands of independent PCR replicates such that not all reactions receive a DNA sequence of interest. The effect of this partitioning process is to reduce the number of molecules in any given reaction and to effectively enrich for sequences of interest. For example, if a sample containing 99 wild type molecules and 1 mutant equates to the mutation being present at 1 in 100 or 1%. Using TaqMan SNP Genotyping Assays in standard real-time PCR mode, the single mutant is lost in a sea of wild type copies. But if we partition the sample, competing wild type sequences in any reaction containing a mutant are reduced, effectively decreasing background noise. If sufficient partitions are used, the reaction wells reach a point where the wild type signal no longer overwhelms the mutant signal. In practice, the total count of each allele, mutant and wild type, can be calculated and a ratio determined since each data point is generated digitally. With the advent of high-throughput next-generation sequencing platforms, screening for cancer-related somatic mutations is becoming commonplace. Digital PCR combined with TaqMan SNP Genotyping Assays offer a highly sensitive and specific solution for the confirmation of these discoveries.