Genomic DNA (gDNA)

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OncoREF

Applied StemCell Inc’s genomic DNA (gDNA) reference standards represent biologically-relevant controls that can be directly incorporated into your sample processing workflows in order to optimize your protocols, evaluate assay sensitivity and specificity, and analyze the impact of workflow changes on downstream analysis. They represent ideal materials for both assay development and routine monitoring of assay performance.

The MAPK Genomic DNA Reference Standards are extracted from ASC’s panel of isogenic MAPK mutation cell lines with 50 recurrent pathway-activating mutations in the EGFR, KRAS and BRAF genes, based on data from the Sanger Institute’s COSMIC database.

Key Features of the gDNA Reference Standards Series:

  • Most comprehensive MAPK mutation panel on the market
  • Well-characterized colorectal cancer cells lines: EGFR (RKO), KRAS (RKO), BRAF (HCT116)
  • Paired, isogenic wild-type cell lines to serve as an ideal control
  • Footprint-free, homozygous mutations
  • Reference cell lines are expanded from single-cells, ensuring maximum homogeneity
  • Available in multiple formats, including slides, scrolls, and full FFPE blocks
Need FFPE standards using your own cell lines?  Contact us for more details.

About the MAPK Pathway

The MAPK signaling pathway encompasses a series of signal transduction events that flow from the engagement of EGFR at the outer cell membrane, through KRAS, BRAF, MEK, and ERK.  MAPK signaling ultimately results in transcriptional activation of key genes that promote cellular proliferation, survival, differentiation, motility, and angiogenesis[1].  As such, the MAPK pathway is one of the most frequently activated pathways in cancer[2], and several drugs have pharmacogenomics profiles that depend upon the MAPK mutational status[3].

mapk-figure

To learn more about our FFPE Reference Standards and other diagnostic products, WATCH our WEBINAR!

  EGFR Mutations Covered by ASC
  CDS Mutation AA Mutation Mutation ID Mutation State
Exon 18 c.2155G>A p.G719A COSM6252  
c.2155G>T p.G719C COSM6253 Homozygous
c.2156G>C p.G719A COSM6239 Homozygous
         
Exon 19 c.2239_2256del18 p.L747_S752delLREATS   Homozygous
c.2240_2257del18 p.L747_P753>S   Homozygous
c.2238_2255del18  p.E746_S752>D   Homozygous
c.2239_2258>CA p.L747_P753>Q   Homozygous
c.2237_2254del18 p.E746_S752>A   Homozygous
c.2237_2255>T p.E746_S752>V   Homozygous
c.2236_2253del18 p.E746_T751delELREAT   Homozygous
c.2237_2251del15 p.E746_T751>A   Homozygous
c.2239_2253del15 p.L747_T751delLREAT   Homozygous
c.2240_2254del15 p.L747_T751delLREAT   Homozygous
c.2237_2252>T p.E746_T751>V   Homozygous
c.2235_2252>AAT p.E746_T751>I   Homozygous
c.2233_2247del15 p.K745_E749delKELRE   Homozygous
c.2235_2249del15 p.E746_A750delELREA   Homozygous
c.2235_2250del15 p.E746_A750delELREA   Homozygous
c.2239_2251>C p.L747_T751>P   Homozygous
c.2239_2251>C p.L747_T751>S   Homozygous
c.2238_2252>GCA p.L747_T751>Q   Homozygous
c.2239_2248TTAAGAGAAG>C p.L747_A750>P   Homozygous
c.2238_2248>GC p.L747_A750>P   Homozygous
c.2239_2247del9 p.L747_E749delLRE   Homozygous
c.2235_2248>AATTC p.E746_A750>IP   Homozygous
         
Exon 20 c.2303G>T p.S768I   Homozygous
c.2307_2308insGCCAGCGTG p.V769_D770insASV   Homozygous
c.2310_2311insGGT p.D770_N771insG   Homozygous
c.2319_2320insCAC p.H773_V774insH   Homozygous
c.2369C>T p.T790M   Homozygous
         
Exon 21 c.2573T>G p.L858R    Homozygous
c.2582T>A p.L861Q    Homozygous
  KRAS Mutations Covered by ASC
  CDS Mutation AA Mutation Mutation ID Mutation State
Exon 2 c.34G>T  p.G12C  COSM516   
c.34G>A p.G12S COSM517  
c.34G>C p.G12R COSM518  
c.35G>T p.G12V COSM520 Homozygous
c.35G>A p.G12D COSM521  
c.35G>C p.G12A COSM522  
c.37G>T  p.G13C COSM527 Homozygous
c.37G>A p.G13S COSM528 Homozygous
c.37G>C p.G13R COSM529 Homozygous
c.38G>A p.G13D COSM532 Homozygous
c.38G>C p.G13A COSM533  
c.38G>T p.G13V COSM534 Homozygous
         
Exon 15 c.1798_1799GT>AA p.V600K COSM473 Homozygous
c.1798_1799GT>AG p.V600R COSM474 Homozygous
c.1799_1800TG>AA p.V600E COSM475 Homozygous 
c.1799T>A p.V600E COSM476 Homozygous
c.1799_1800TG>AT p.V600D COSM477 Homozygous
c.1798G>A p.V600M COSM1130 Homozygous
c.1799T>G p.V600G COSM6147 Homozygous
 
Technical Details

Consistent Source of Biorelevant Specimens

Genomic DNA reference standards perform efficiently as reference material for qPCR applications:

  • Ability to detect mutations using qPCR platforms
  • Amplification efficiency close to 1
  • Idea for qPCR assay development and validation
TECHNICAL-MAPK-genomicDNA-2-qPCR-AJH

Support Materials

ONCOREF™ Reference Standards: Application of CRISPR/Cas9 to the Generation of Isogenic Cell Lines and Reference Materials (October 2016)

2016-Oncoref-webinar

 

 

 

 

 

 

 

 

 

CRISPR/Cas9 is rapidly enabling the development of new tools for enhancing our understanding oncogenic mutations in cancer. In order to aid in advancing cancer diagnosis and treatment, Applied StemCell has recently engineered a series of 40 isogenic cell lines that feature diverse mutations in the MAPK pathway. These mutant lines are available as isogenic pairs for applications in lead compound discovery, or as FFPE and nucleic acid reference materials for assay development. This webinar will focus on ASC’s efforts in developing these research tools, as well as applications of the materials for the advancement of cancer research.

Highlights of this talk:

  • Overview of molecular reference materials 
  • Workflow and QC for ONCOREF™ cell line generation
  • Advantages of CRISPR-engineered molecular reference standards
  • Applications of reference materials in assay development
  • Q & A
FAQ

1. How are your cell lines generated?

Answer: ASC uses CRISPR/Cas9 technology to enable precision genome editing in its MAPK mutation panel series.  This technology allows for footprint-free gene modification, meaning that you don’t have to worry about the presence of selection markers or other genomic footprints during the development of your assay technologies.

2. How do you validate mutational status in your cell lines? 

Answer: All of our MAPK mutation panel cell lines are expanded from single clones.  This ensures maximum homogeneity of the genetic profile.  After clonal expansion, we confirm the mutational status of the cell line using Sanger sequencing.

3. How do you assess the quality of the gDNA?

         Answer: We use agarose gel electrophoresis and qunatitiative PCR of GAPDH locus.  

4. How do you quantify the gDNA (single mutation)?

         Answer: We use spectrophotometry (A260).  

5.  What does COSMIC ID stands for?

         Answer: COSMIC is an acronym for Catalogue of Somatic Mutations in Cancer.  

5. Do you offer mixtures of mutant and wild-type DNA?

Answer: We are currently working with collaborators to develop these products.  Please inquire for additional information.

References
  1. Akinleye A, Furqan M, Mukhi N, Ravella P, Liu D (2013) MEK and the inhibitors: from bench to bedside. J. Hematol. Oncol. 6, 1–11
  2. Santarpia L, Lippman SM, El-Naggar AK (2012) Targeting the MAPK–RAS–RAF signaling pathway in cancer therapy, Expert Opin. Ther. Targets 16, 103–119
  3. Relling MV, Evans WE (2015) Pharmacogenomics in the clinic, Nature 526, 343–350
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