Microchimica Acta

, Volume 181, Issue 13–14, pp 1681–1688 | Cite as

Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment

  • Oliver Strohmeier
  • Silke Laßmann
  • Bianca Riedel
  • Daniel Mark
  • Günter Roth
  • Martin Werner
  • Roland Zengerle
  • Felix von Stetten
Original Paper


Point Mutations on the Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified as an important predictive biomarker for response to cancer therapy targeting the epidermal growth factor receptor. KRAS mutations are prevalent in up to 40 % of all colorectal carcinomas, and routinely conducted KRAS genotyping is becoming mandatory to predict therapy success and to reduce therapy costs. We report a low-cost, disposable and ready-to-use centrifugal microfluidic cartridge (termed GeneSlice) containing preloaded primers and probes. The GeneSlice cartridge enables the parallel detection of the seven most relevant KRAS point mutations by allele-specific real-time PCR. It represents a cost effective alternative to dideoxy-sequencing with a faster time-to-result (~ 2 h versus up to 20 h in case of dd-sequencing). Microfluidic processing of the GeneSlice along with allele-specific amplification and real-time detection are conducted in a slightly modified, commercially available PCR thermocycler. Intra-chip standard deviation of Cq values on the GeneSlices is negligible (GeneSlice 1: Cq, = 0.13; GeneSlice 2: Cq, = 0.26). In 23 of 24 experiments, the data for genotyping 6 cancer cell lines (n = 4 per cell line) agreed with dd-sequencing. Additionally, DNA derived from microdissected formalin-fixed and paraffin embedded colorectal carcinomas of two cases was genotyped correctly and reproducibly (n = 3 per patient; one GeneSlice excluded from evaluation). The GeneSlice therefore clearly demonstrated the potential to become a valuable tool for routine diagnostics of KRAS mutations by reducing costs and hands-on time.


Photograph of a centrifugal microfluidic cartridge “GeneSlice” for multiplex genotyping of KRAS point mutations from tumor cell DNA by allele-specific real-time PCR. Information about the mutation status is required to predict success of state-of-the-art cancer therapy with antibodies


Centrifugal microfluidics GeneSlice Allele-specific PCR Lab-on-a-Chip KRAS 



We gratefully acknowledge financial support from the Baden-Württemberg Foundation for funding this project (“Amplidisk” - MST II – 17). Additionally, we want to thank Dominique Kosse and the team of HSG-IMIT Microfluidic Design & Foundry Service for fabrication of the GeneSlices. S. Laßmann, B. Riedel and M. Werner greatly acknowledge the support by Dr. Sandra Lass, Institute of Pathology, University Medical Center, Freiburg for excellent coordinative support of this project.


  1. 1.
    Lang AH, Drexel H, Geller-Rhomberg S, Start N, Winder T, Geiger K, Muendlein A (2011) Optimized Allele-Specific Real-Time PCR assays for the Detection of Common Mutations in KRAS and BRAF. JMD 13:23–27CrossRefGoogle Scholar
  2. 2.
    Laßmann S, Werner M (2012) Predictive pathology in routine diagnostics of solid tumors. Histol Histopathol 27:289–296Google Scholar
  3. 3.
    Stintzing S, Heinemann V, Moosmann N, Hiddemann W, Jung A, Kirchner T (2009) The treatment of colorectal carcinoma with monoclonal antibodies: the importance of KRAS mutation analysis and EGFR status. Deutsches Ärzteblatt 106:202–206Google Scholar
  4. 4.
    Bos JL, Fearon ER, Hamilton SR, Verlaandevries M, Vanboom JH, Vandereb AJ, Vogelstein B (1987) Prevalence of Ras Gene-Mutations in Human Colorectal Cancers. Nature 327:293–297CrossRefGoogle Scholar
  5. 5.
    van Krieken JH et al (2008) KRAS Mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program. Virchows Arch 453:417–431CrossRefGoogle Scholar
  6. 6.
    Medscape Medical News, “GICS 2009: Huge Cost Savings From KRAS Testing in Metastatic Colorectal Cancer”, (Accessed: 23.01.2013)
  7. 7.
    Brockman W, Alvarez P, Young S, Garber M, Giannoukos G, Lee WL, Russ C, Lander ES, Nusbaum C, Jaffe DB (2008) Quality scores and SNP detection in sequencing-by-synthesis systems. Genome Research 18:763–770CrossRefGoogle Scholar
  8. 8.
    Frazer KA et al (2007) A second generation human haplotype map of over 3.1 million SNPs. Nature 449:851–861CrossRefGoogle Scholar
  9. 9.
    Khanna M, Park P, Zirvi M, Cao WG, Picon A, Day J, Paty P, Barany F (1999) Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors. Oncogene 18:27–38CrossRefGoogle Scholar
  10. 10.
    Hacia JG et al (1999) Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays. Nat Genet 22:164–167CrossRefGoogle Scholar
  11. 11.
    Sohni YR, Cerhan JR, O’Kane D (2003) Microarray and microfluidic methodology for genotyping cytokine gene polymorphisms. Hum Immunol 64:990–997CrossRefGoogle Scholar
  12. 12.
    Kan CW, Fredlake CP, Doherty EA, Barron AE (2004) DNA sequencing and genotyping in miniaturized electrophoresis systems. Electrophoresis 25:3564–3588CrossRefGoogle Scholar
  13. 13.
    Krypuy M, Newnham GM, Thomas DM, Conron M, Dobrovic A (2006) High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer 6:295CrossRefGoogle Scholar
  14. 14.
    Manz A, Graber N, Widmer HM (1990) Miniaturized Total Chemical-Analysis Systems - A Novel Concept for Chemical Sensing. Sensor Actuat B-Chem 1:244–248CrossRefGoogle Scholar
  15. 15.
    Dittrich PS, Manz A (2006) Lab-on-a-chip: microfluidics in drug discovery. Nat Rev Drug Discov 5:210–218CrossRefGoogle Scholar
  16. 16.
    Erickson D, Liu XZ, Venditti R, Li DQ, Krull UJ (2005) Electrokinetically based approach for single nucleotide polymorphism discrimination using a microfluidic device. Anal Chem 77:4000–4007CrossRefGoogle Scholar
  17. 17.
    Hatakeyama K et al (2009) Microfluidic device using chemiluminescence and a DNA-arrayed thin film transistor photosensor for single nucleotide polymorphism genotyping of PCR amplicons from whole blood. Lab Chip 9:1052–1058CrossRefGoogle Scholar
  18. 18.
    Chowdhury J et al (2007) Microfluidic Platform for Single Nucleotide Polymorphism Genotyping of the Thiopurine S-Methyltransferase Gene to Evaluate Risk for Adverse Drug Events. J Mol Diagn 9:521–529CrossRefGoogle Scholar
  19. 19.
    Pekin D et al., (2012), Quantitative detection of circulating tumor DNA in plasma samples by droplet digital PCR, Proc. of μTAS, 169–171Google Scholar
  20. 20.
    Taly V, Pekin D, El Abed A, Laurent-Puig P (2012) Detecting biomarkers with microdroplet technology. Trends Mol Med 18:405–416CrossRefGoogle Scholar
  21. 21.
    Pekin D et al (2011) Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Lab Chip 11:2156–2166CrossRefGoogle Scholar
  22. 22.
    Lutz S et al (2010) Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification RPA. Lab Chip 10:887–893CrossRefGoogle Scholar
  23. 23.
    Focke M et al (2010) Microstructuring of polymer films for sensitive genotyping by real-time PCR on a centrifugal microfluidic platform. Lab Chip 10:2519–2526CrossRefGoogle Scholar
  24. 24.
    Focke M, Stumpf F, Roth G, Zengerle R, von Stetten F (2010) Centrifugal microfluidic system for primary amplification and secondary real-time PCR. Lab Chip 10:3210–3212CrossRefGoogle Scholar
  25. 25.
    Focke M, Kosse D, Al-Bamerni D, Lutz S, Müller C, Reinecke H, Zengerle R, von Stetten F (2011) Microthermoforming of microfluidic substrates by soft lithography (μTSL): optimization using design of experiments. J Micromech Microeng 21:115002CrossRefGoogle Scholar
  26. 26.
    Herz C et al (2012) Occurrence of Aurora A positive multipolar mitoses in distinct molecular classes of colorectal carcinomas and effect of Aurora A inhibition. Mol Carcinog 51:696–710CrossRefGoogle Scholar
  27. 27.
    Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF (1989) Analysis of Any Point Mutation in Dna - the Amplification Refractory Mutation System (Arms). Nucleic Acid Res 17:2503–2516CrossRefGoogle Scholar
  28. 28.
    Mark D, Metz T, Haeberle S, Lutz S, Ducrée J, Zengerle R, von Stetten F (2009) Centrifugo-Pneumatic Valve for Metering of Highly Wetting Liquids on Centrifugal Microfluidic Platforms. Lab Chip 9:3599–3603CrossRefGoogle Scholar
  29. 29.
    SABiosciences, a QIAGEN Company; qBiomarker Somatic Mutation PCR Array: Human KRAS Gene (Accessed: 26.03.2013)
  30. 30.
    Neumann J, Eberhart E, Kirchner T, Jung A (2009) Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer. Pathol Res Pract 205:858–862CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Oliver Strohmeier
    • 1
    • 2
  • Silke Laßmann
    • 3
    • 4
    • 5
    • 6
  • Bianca Riedel
    • 3
    • 6
  • Daniel Mark
    • 1
  • Günter Roth
    • 2
    • 4
  • Martin Werner
    • 3
    • 5
    • 6
  • Roland Zengerle
    • 1
    • 2
    • 4
  • Felix von Stetten
    • 1
    • 2
  1. 1.HSG-IMIT - Institut für Mikro- und InformationstechnikFreiburgGermany
  2. 2.Laboratory for MEMS Applications, IMTEK - Department of Microsystems EngineeringUniversity of FreiburgFreiburgGermany
  3. 3.Institute of PathologyUniversity Medical CenterFreiburgGermany
  4. 4.BIOSS – Centre for Biological Signalling StudiesUniversity of FreiburgFreiburgGermany
  5. 5.Comprehensive Cancer Center FreiburgFreiburgGermany
  6. 6.German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ)HeidelbergGermany

Personalised recommendations