{"id":7024,"date":"2015-05-22T09:35:30","date_gmt":"2015-05-22T09:35:30","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/?p=7024"},"modified":"2020-05-28T19:14:23","modified_gmt":"2020-05-28T19:14:23","slug":"go-digital-pcr-pt-5-rare-mutations","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/go-digital-pcr-pt-5-rare-mutations\/","title":{"rendered":"Go Digital PCR: Pt 5 \u2013 Rare Mutations"},"content":{"rendered":"

Go Digital PCR: Pt 5 \u2013 Rare Mutations <\/em><\/strong>is the fifth segment in our six-part series introducing you to digital PCR (dPCR)<\/a> and how it is capable of detecting and quantifying rare mutations for low-prevalence targets in cancer research samples.<\/p>\n

Rare mutation detection is a powerful tool in cancer research<\/strong><\/p>\n

In oncologic research, rare mutation detection<\/a> among a large population of wild type cells poses a significant challenge. The accumulation of mutations in oncogenes or tumor suppressor genes is an important aspect of tumor formation. Acquisition of these mutations in a tiny subset of somatic cells can be sufficient for cancer initiation or progression.<\/p>\n

Common SNP genotyping technologies, such as capillary electrophoresis<\/a> or real-time qPCR, are effective at detecting mutant cells with prevalence no lower than about 20% \u2013 approximately one in five cells. But because these mutations are usually present only in a very small number of cells, they require an assay that delivers high signal-to-noise and low false-positive rates. dPCR has that capability. By subdividing a sample into thousands of individual PCR replicates, the total number of molecules in any given reaction is greatly reduced, effectively enriching for the sequences of interest and diluting out the wild type background.<\/p>\n

There are also times where dPCR is useful in detecting very low quantities of nucleic acids in heterogeneous samples, i.e., allele detection. In tumor samples, for example, dPCR can be used to determine whether a small percentage of cells have a mutant allele within an oncogene.<\/p>\n

Researchers are now able to detect low-frequency mutations by combining TaqMan\u2122 fluorogenic 5\u2019 nuclease chemistry with dPCR methodology using the QuantStudio\u2122 3D Digital PCR System<\/a>.\u00a0 Together with TaqMan\u00ae<\/sup> SNP Genotyping Assays<\/a>, the resulting QuantStudio\u2122 3D rare mutation analysis solution<\/a> helps achieve the high levels of performance required to detect and absolutely quantify mutations present at a very low frequency.<\/p>\n

Enables rare mutation detection in just hours with the QuantStudio\u2122 3D Digital PCR System <\/strong><\/p>\n

The QuantStudio\u2122 3D Digital PCR System is a simple and affordable dPCR platform for enabling the detection and quantification of rare mutations for low-prevalence targets in cancer research samples. The QuantStudio\u2122 3D leverages high-density nanofluidic chip technology to partition a sample into as many as 20,000 independent reaction wells. Comprised of consistently sized wells etched in a solid silicon substrate, the QuantStudio\u2122 3D Digital PCR Chip enables straightforward and robust sample partitioning that allows thousands of data points to be analyzed per run. The highly controlled sample loading and low reaction dropout combine to ensure higher precision for absolute quantification.<\/p>\n

Key advantages of using the QuantStudio\u2122 3D rare mutation analysis solution include:<\/p>\n

\u2022\u00a0 Enables optimized dPCR performance<\/p>\n

\u2022\u00a0 38 wet lab-validated TaqMan\u00ae<\/sup> SNP Genotyping Assays targeting 60 somatic mutations<\/p>\n

\u2022\u00a0 Helps detect and quantify rare mutant prevalence as low as 0.1%<\/p>\n

\u2022\u00a0 Single-tube format includes wild type and mutant alleles, with small and large reaction options<\/p>\n

\u2022\u00a0 Advanced algorithms help streamline quantification of rare mutations<\/p>\n

Greater precision with wet lab-validated TaqMan\u2122 SNP Genotyping Assays
\n<\/strong>Dr. Iain Russell, Sr. Product Manager for TaqMan\u00ae<\/sup> Assays<\/a>, spoke of the most recent QuantStudio\u2122 3D enhancements to benefit rare mutation analysis in an interview at the American Society for Human Genetics (ASHG) 2014 annual meeting. Specifically, Dr. Russell described the QuantStudio\u2122 3D rare mutation analysis solution\u2019s subset of 38 wet lab\u2013validated TaqMan\u00ae<\/sup> SNP Genotyping Assays for use in detecting and quantifying rare somatic mutations, e.g., EGFR, BRAF, KRAS, PIK3CA, and JAK2. The assays are wet lab-validated specifically for our QuantStudio\u2122 3D Digital PCR System and allow you to quantify rare mutants at a prevalence as low as 0.1%.<\/p>\n

\"dpcr5\"<\/p>\n

CAPTION:<\/strong><\/span> This is an example of dPCR quantification of a rare KRAS<\/em> allele in which a KRAS G12V mutation was detected. Each sample represents a fraction of mutant plasmid to wild-type genomic DNA. The 0.1% target represents mutant genomic DNA from a mutant cell line in a wild-type genomic DNA background. All samples were analyzed using QuantStudio\u2122 3D AnalysisSuite\u2122<\/sup> Cloud Software with the relative quantification module. (A<\/strong>)Targeted mutation rate: 10%. Mutation detection rate: 10.384% with a confidence interval of 8.235\u201313.076%. (B<\/strong>) Targeted mutation rate: 1%. Mutation detection rate: 1.393% with a confidence interval of 0.772\u20132.507%. (C<\/strong>) Targeted mutation rate: 0.1%. Mutation detection rate: 0.142% with a confidence interval of 0.064\u20130.310%. (D<\/strong>) Wild type control sample. <\/span><\/p>\n

Target selection for the wet lab\u2013validated SNP genotyping assays was based on frequency of occurrence as well as targets that exist on the Ion AmpliSeq\u2122<\/sup> Cancer Hotspot Panel.<\/a>\u00a0 The assays are single-tube assays that contain primers and a probe for both wild type and mutant alleles, and are available in 12- and 450-reaction sizes<\/a>.<\/p>\n

Helps you perform absolute quantification with confidence <\/strong><\/p>\n

QuantStudio\u2122 3D AnalysisSuite\u2122<\/sup> Cloud Software<\/a>, allows users to quantify the percentage of mutation in their samples from the data collected on the QuantStudio\u2122 3D Digital PCR System.<\/p>\n

In applications involving rare allele detection, AnalysisSuite\u2122<\/sup> Software sequesters the results of individual PCR reactions into four different quadrants. The upper left quadrant (blue dots) is almost exclusively allele A, while the lower right quadrant is almost exclusively allele B (red dots).\u00a0 The green spots are the wells with both allele A and B, and the yellow dots are unamplified.<\/p>\n

\"dpcr5a\"<\/a><\/p>\n

* These %s were calculated by Excel using data from AnalysisSuite\u2122 Software<\/p>\n

To illustrate this a collaborator Dr. Antonio Jimenez-Velasco (Carlos Haya Hospital, Malaga Spain) sent Thermo Fisher Scientific researchers leukemia samples post bone marrow transplant. Assays were selected where the bone marrow donors possessed a null allele of a locus and the bone marrow recipients samples possessed an insertion allele in the same locus, facilitating discrimination. Blood samples were collected from the donors and recipients before transplant and various times after transplant. The presence of the two loci in the samples was quantified by dPCR.<\/p>\n

The graph shows the results from one recipient sample. Before the bone marrow transplant \u2013 Pre-SCT \u2013 the genotype of the circulating cells was 100% of the host’s genotype. The donor, shown in the next position had about 0.1% of the recipient\u2019s allele. Over time, you can see that after 62 days, 147 days, and 174 days, there are low levels of the recipient sample\u2019s original genotype. However, it starts to come up after 192 days, indicating that the recipient sample\u2019s cells were repopulating the bone marrow. Error bars represent a calculated 95% confidence interval.<\/p>\n

The collaborator was still unable to detect the chimerism before 192 days by traditional PCR. dPCR was more sensitive and picked up the chimerism (reappearance of original allele) earlier than traditional PCR.<\/p>\n

Note: Mixed chimerism occurs in a leukemia sample when both the recipient and donor cell samples are present in bone marrow. It\u2019s routinely analyzed after stem cell transplantation.<\/p>\n

Conclusion<\/strong><\/p>\n

I hope this primer on dPCR and rare mutation detection and quantification has been enlightening and that the links herein will help your understanding of dPCR even more. To recap, the QuantStudio\u2122 3D rare mutation analysis solution offers your lab:<\/p>\n

\u2022\u00a0 Optimized digital PCR performance enhanced by 38 wet lab\u2013validated TaqMan\u00ae<\/sup> SNP Genotyping Assays targeting 60 somatic mutations<\/p>\n

\u2022\u00a0 High sensitivity to detect and quantify rare mutation prevalence as low as 0.1%<\/p>\n

\u2022\u00a0 Cost-effective and convenient single-tube format that includes wild type and mutant alleles with small and large reaction options<\/p>\n

\u2022\u00a0 Streamlined analyses with advanced algorithms for better quantification of rare mutations<\/p>\n

Learn more about dPCR and rare mutation detection and quantification<\/strong><\/p>\n

Learn more about how the QuantStudio\u2122 3D Digital PCR System<\/a> enables detection and quantification of rare mutations<\/a> for low-prevalence targets in cancer research samples. For more on the latest TaqMan\u00ae<\/sup> SNP Genotyping Assays available for rare mutation detection, take a moment to watch this recent video interview with Dr. Iain Russell, Life Technologies\u2019 Sr. Product Manager for Digital PCR Technology at ASHG 2014: Rare Mutation Detection w\/Digital PCR<\/em> <\/a><\/p>\n

In the world of webinars, we offer you several ways to expand your awareness of dPCR and rare mutation detection and quantification.<\/p>\n

First up is Expanding the PCR Tool Box with Digital PCR: A review of absolute quantification and mutation detection applications using the QuantStudio\u2122 3D Digital PCR System.<\/em><\/a> Steve Jackson, PhD, Thermo Fisher Scientific’s Associate Director of Product Applications in the Genetic Analysis division is the speaker. You\u2019ll hear how digital PCR expands the application boundaries of traditional real-time PCR and see real world examples of how you can go beyond measuring Ct to detecting individual molecules, including oncogenic alleles in FFPE cancer specimens, and gaining additional sensitivity and precision for a variety of experiments.<\/p>\n

Next, if you\u2019re entirely new to dPCR, Dr. Iain Russell, Sr. Life Technologies\u2019 Product Manager for Digital PCR Technology, can help you wrap your head around the basics with his webinar: Introduction to Digital PCR \u2013 Sensitive, precise, absolute quantification with chip-based digital PCR<\/a> <\/em>Listen in as he covers the benefits of digital PCR, how it works, and how to use it for applications that require increased sensitivity and precision. You\u2019ll also learn how the QuantStudio\u2122 3D Digital PCR System employs a chip-based technology to perform simple and robust digital PCR.<\/p>\n

 <\/p>\n

Read all of the Let’s Go Digital PCR Series:<\/p>\n

Go Digital PCR: Pt 1 \u2013 Next-Gen Quantification<\/a><\/p>\n

Go Digital PCR: Pt 2 \u2013 QuantStudio\u2122 3D<\/a><\/p>\n

Go Digital PCR: Pt 3 – Copy Number Variation<\/a><\/p>\n

Go Digital PCR: Pt 4 \u2013 NGS Libraries<\/a><\/p>\n

Go Digital PCR: Pt 5 \u2013 Rare Mutations<\/a><\/p>\n

Go Digital PCR: Pt 6 – Emerging Analyses<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"

Go Digital PCR: Pt 5 \u2013 Rare Mutations is the fifth segment in our six-part series introducing you to digital PCR (dPCR) and how it is capable of detecting and quantifying rare mutations for low-prevalence targets in cancer research samples. Rare mutation detection is a powerful tool in cancer research In oncologic research, rare mutation<\/p>\n","protected":false},"author":120,"featured_media":7039,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[108,101],"tags":[17,189,57,43],"division":[],"class_list":{"0":"post-7024","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-lab-tips-and-tricks","8":"category-product-news","9":"tag-applied-biosystems","10":"tag-bioinformatics-software","11":"tag-digital-pcr","12":"tag-pcr","13":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"\nGo Digital PCR: Pt 5 \u2013 Rare Mutations - Behind the Bench<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.thermofisher.com\/blog\/behindthebench\/go-digital-pcr-pt-5-rare-mutations\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Go Digital PCR: Pt 5 \u2013 Rare Mutations\" \/>\n<meta property=\"og:description\" content=\"Go Digital PCR: Pt 5 \u2013 Rare Mutations is the fifth segment in our six-part series introducing you to digital PCR (dPCR) and how it is capable of detecting and quantifying rare mutations for low-prevalence targets in cancer research samples. 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