Spandidos Publications Logo
  • About
    • About Spandidos
    • Aims and Scopes
    • Abstracting and Indexing
    • Editorial Policies
    • Reprints and Permissions
    • Job Opportunities
    • Terms and Conditions
    • Contact
  • Journals
    • All Journals
    • Oncology Letters
      • Oncology Letters
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • International Journal of Oncology
      • International Journal of Oncology
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Molecular and Clinical Oncology
      • Molecular and Clinical Oncology
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Experimental and Therapeutic Medicine
      • Experimental and Therapeutic Medicine
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • International Journal of Molecular Medicine
      • International Journal of Molecular Medicine
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Biomedical Reports
      • Biomedical Reports
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Oncology Reports
      • Oncology Reports
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Molecular Medicine Reports
      • Molecular Medicine Reports
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • World Academy of Sciences Journal
      • World Academy of Sciences Journal
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • International Journal of Functional Nutrition
      • International Journal of Functional Nutrition
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • International Journal of Epigenetics
      • International Journal of Epigenetics
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Medicine International
      • Medicine International
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
  • Articles
  • Information
    • Information for Authors
    • Information for Reviewers
    • Information for Librarians
    • Information for Advertisers
    • Conferences
  • Language Editing
Spandidos Publications Logo
  • About
    • About Spandidos
    • Aims and Scopes
    • Abstracting and Indexing
    • Editorial Policies
    • Reprints and Permissions
    • Job Opportunities
    • Terms and Conditions
    • Contact
  • Journals
    • All Journals
    • Biomedical Reports
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Experimental and Therapeutic Medicine
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • International Journal of Epigenetics
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • International Journal of Functional Nutrition
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • International Journal of Molecular Medicine
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • International Journal of Oncology
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Medicine International
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Molecular and Clinical Oncology
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Molecular Medicine Reports
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Oncology Letters
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • Oncology Reports
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
    • World Academy of Sciences Journal
      • Information for Authors
      • Editorial Policies
      • Editorial Board
      • Aims and Scope
      • Abstracting and Indexing
      • Bibliographic Information
      • Archive
  • Articles
  • Information
    • For Authors
    • For Reviewers
    • For Librarians
    • For Advertisers
    • Conferences
  • Language Editing
Login Register Submit
  • This site uses cookies
  • You can change your cookie settings at any time by following the instructions in our Cookie Policy. To find out more, you may read our Privacy Policy.

    I agree
Search articles by DOI, keyword, author or affiliation
Search
Advanced Search
presentation
Molecular Medicine Reports
Join Editorial Board Propose a Special Issue
Print ISSN: 1791-2997 Online ISSN: 1791-3004
Journal Cover
September-2017 Volume 16 Issue 3

Full Size Image

Sign up for eToc alerts
Recommend to Library

Journals

International Journal of Molecular Medicine

International Journal of Molecular Medicine

International Journal of Molecular Medicine is an international journal devoted to molecular mechanisms of human disease.

International Journal of Oncology

International Journal of Oncology

International Journal of Oncology is an international journal devoted to oncology research and cancer treatment.

Molecular Medicine Reports

Molecular Medicine Reports

Covers molecular medicine topics such as pharmacology, pathology, genetics, neuroscience, infectious diseases, molecular cardiology, and molecular surgery.

Oncology Reports

Oncology Reports

Oncology Reports is an international journal devoted to fundamental and applied research in Oncology.

Experimental and Therapeutic Medicine

Experimental and Therapeutic Medicine

Experimental and Therapeutic Medicine is an international journal devoted to laboratory and clinical medicine.

Oncology Letters

Oncology Letters

Oncology Letters is an international journal devoted to Experimental and Clinical Oncology.

Biomedical Reports

Biomedical Reports

Explores a wide range of biological and medical fields, including pharmacology, genetics, microbiology, neuroscience, and molecular cardiology.

Molecular and Clinical Oncology

Molecular and Clinical Oncology

International journal addressing all aspects of oncology research, from tumorigenesis and oncogenes to chemotherapy and metastasis.

World Academy of Sciences Journal

World Academy of Sciences Journal

Multidisciplinary open-access journal spanning biochemistry, genetics, neuroscience, environmental health, and synthetic biology.

International Journal of Functional Nutrition

International Journal of Functional Nutrition

Open-access journal combining biochemistry, pharmacology, immunology, and genetics to advance health through functional nutrition.

International Journal of Epigenetics

International Journal of Epigenetics

Publishes open-access research on using epigenetics to advance understanding and treatment of human disease.

Medicine International

Medicine International

An International Open Access Journal Devoted to General Medicine.

Journal Cover
September-2017 Volume 16 Issue 3

Full Size Image

Sign up for eToc alerts
Recommend to Library

  • Article
  • Citations
    • Cite This Article
    • Download Citation
    • Create Citation Alert
    • Remove Citation Alert
    • Cited By
  • Similar Articles
    • Related Articles (in Spandidos Publications)
    • Similar Articles (Google Scholar)
    • Similar Articles (PubMed)
  • Download PDF
  • Download XML
  • View XML
Article Open Access

Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS

  • Authors:
    • Jia Peng
    • Kun Wei
    • Xiang Zhao
    • Ke Yang
    • Huan Wang
    • Yang Zhang
    • Mei Guo
    • Jing He
    • Haiyan Wu
    • Yongchuan Li
    • Na Zhao
    • Qing Huang
    • Weiling Fu
  • View Affiliations / Copyright

    Affiliations: Department of Laboratory Medicine, Southwest Hospital, The Third Military Medical University, Chongqing 400038, P.R. China
    Copyright: © Peng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Pages: 2726-2732
    |
    Published online on: June 29, 2017
       https://doi.org/10.3892/mmr.2017.6883
  • Expand metrics +
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )
Cited By (CrossRef): 0 citations Loading Articles...

This article is mentioned in:



Abstract

Mutant KRAS proto‑oncogene GTPase (KRAS) serves an important role in predicting the development, diagnosis, treatment and efficacy of targeted drug therapies for colorectal cancer. To improve the detection efficacy of trace amount of mutant KRAS, the locked nucleic acid‑based method was modified in the present study. Internal competitive amplification fragments were used to improve the inhibition of wild‑type KRAS with a wild‑type blocking (WTB) probe and specifically amplify the trace amounts of mutant KRAS. The modified method, quantitative clamp‑based polymerase chain reaction technology using WTB coupled with internal competitive reference to enhance the amplification specificity, named WIRE‑PCR, completely blocked the amplification of wild‑type KRAS in 50‑150 ng DNA templates. The added internal competitive amplified fragments were amplified together with the target gene, which were used to reduce base mismatch due to the high number of cycles in PCR and quantify the total amount of DNA. The results demonstrated that WIRE‑PCR facilitated the detection of mutated alleles at a single molecular level. In the colorectal biopsies from 50 patients with suspected colorectal cancer, 18 cases (36%) contained mutant KRAS, and the amount of mutant DNA accounted for 18.6‑64.2% of the total DNA. WIRE‑PCR is a simple, rapid and low‑cost quantitative analysis method for the detection of trace amounts of the mutant KRAS.
View Figures

Figure 1

Figure 2

Figure 3

Figure 4

View References

1 

Ferlay J, Shin HR, Bray F, Forman D, Mathers C and Parkin DM: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 127:2893–2917. 2010. View Article : Google Scholar : PubMed/NCBI

2 

Siegel R, Naishadham D and Jemal A: Cancer statistics, 2013. CA Cancer J Clin. 63:11–30. 2013. View Article : Google Scholar : PubMed/NCBI

3 

Venook AP: Epidermal growth factor receptor-targeted treatment for advanced colorectal carcinoma. Cancer. 103:2435–2446. 2005. View Article : Google Scholar : PubMed/NCBI

4 

Wadlow RC, Hezel AF, Abrams TA, Blaszkowsky LS, Fuchs CS, Kulke MH, Kwak EL, Meyerhardt JA, Ryan DP, Szymonifka J, et al: Panitumumab in patients with KRAS wild-type colorectal cancer after progression on cetuximab. Oncologist. 17:142012. View Article : Google Scholar : PubMed/NCBI

5 

Karapetis CS, Khambata-Ford S, Jonker DJ, O'Callaghan CJ, Tu D, Tebbutt NC, Simes RJ, Chalchal H, Shapiro JD, Robitaille S, et al: K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med. 359:1757–1765. 2008. View Article : Google Scholar : PubMed/NCBI

6 

Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ, Juan T, Sikorski R, Suggs S, Radinsky R, et al: Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 26:1626–1634. 2008. View Article : Google Scholar : PubMed/NCBI

7 

Kranenburg O: The KRAS oncogene: Past, present, and future. Biochim Biophys Acta. 1756:81–82. 2005.PubMed/NCBI

8 

De Roock W, Jonker DJ, Di Nicolantonio F, Sartore-Bianchi A, Tu D, Siena S, Lamba S, Arena S, Frattini M, Piessevaux H, et al: Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA. 304:1812–1820. 2010. View Article : Google Scholar : PubMed/NCBI

9 

Anderson SM: Laboratory methods for KRAS mutation analysis. Expert Rev Mol Diagn. 11:635–642. 2011. View Article : Google Scholar : PubMed/NCBI

10 

Lenz HJ: Testing for RAS mutations in patients with metastatic colorectal cancer. Clin Adv Hematol Oncol. 12:48–49. 2014.PubMed/NCBI

11 

Oldenburg RP, Liu MS and Kolodney MS: Selective amplification of rare mutations using locked nucleic acid oligonucleotides that competitively inhibit primer binding to wild-type DNA. J Invest Dermatol. 128:398–402. 2008. View Article : Google Scholar : PubMed/NCBI

12 

Huang Q, Wang GY, Huang JF, Zhang B and Fu WL: High sensitive mutation analysis on KRAS gene using LNA/DNA chimeras as PCR amplification blockers of wild-type alleles. Mol Cell Probes. 24:376–380. 2010. View Article : Google Scholar : PubMed/NCBI

13 

Dominguez PL and Kolodney MS: Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens. Oncogene. 24:6830–6834. 2005. View Article : Google Scholar : PubMed/NCBI

14 

Huang JF, Zeng DZ, Duan GJ, Shi Y, Deng GH, Xia H, Xu HQ, Zhao N, Fu WL and Huang Q: Single-tubed wild-type blocking quantitative pcr detection assay for the sensitive detection of codon 12 and 13 KRAS mutations. PLoS One. 10:e1456982015. View Article : Google Scholar

15 

Di Giusto DA and King GC: Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: Implications for genotyping assays. Nucleic Acids Res. 32:e322004. View Article : Google Scholar : PubMed/NCBI

16 

You Y, Moreira BG, Behlke MA and Owczarzy R: Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res. 34:e602006. View Article : Google Scholar : PubMed/NCBI

17 

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, et al: The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 55:611–622. 2009. View Article : Google Scholar : PubMed/NCBI

18 

Custodio A and Feliu J: Prognostic and predictive biomarkers for epidermal growth factor receptor-targeted therapy in colorectal cancer: Beyond KRAS mutations. Crit Rev Oncol Hematol. 85:45–81. 2013. View Article : Google Scholar : PubMed/NCBI

19 

Kimura T, Okamoto K, Miyamoto H, Kimura M, Kitamura S, Takenaka H, Muguruma N, Okahisa T, Aoyagi E, Kajimoto M, et al: Clinical benefit of high-sensitivity KRAS mutation testing in metastatic colorectal cancer treated with anti-EGFR antibody therapy. Oncology. 82:298–304. 2012. View Article : Google Scholar : PubMed/NCBI

20 

Smit VT, Boot AJ, Smits AM, Fleuren GJ, Cornelisse CJ and Bos JL: KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Res. 16:7773–7782. 1988. View Article : Google Scholar : PubMed/NCBI

21 

Eberhard DA, Johnson BE, Amler LC, Goddard AD, Heldens SL, Herbst RS, Ince WL, Jänne PA, Januario T, Johnson DH, et al: Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol. 23:5900–5909. 2005. View Article : Google Scholar : PubMed/NCBI

22 

Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, Ychou M, Bouché O, Landi B, Louvet C, et al: KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol. 26:374–379. 2008. View Article : Google Scholar : PubMed/NCBI

23 

Itonaga M, Matsuzaki I, Warigaya K, Tamura T, Shimizu Y, Fujimoto M, Kojima F, Ichinose M and Murata S: Novel methodology for rapid detection of KRAS mutation using PNA-LNA mediated loop-mediated isothermal amplification. PLoS One. 11:e1516542016. View Article : Google Scholar

24 

Dono M, Massucco C, Chiara S, Sonaglio C, Mora M, Truini A, Cerruti G, Zoppoli G, Ballestrero A, Truini M, et al: Low percentage of KRAS mutations revealed by locked nucleic acid polymerase chain reaction: Implications for treatment of metastatic colorectal cancer. Mol Med. 18:1519–1526. 2013.PubMed/NCBI

25 

Chen D, Yang Z, Xia H, Huang JF, Zhang Y, Jiang TN, Wang GY, Chuai ZR, Fu WL and Huang Q: Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube. PLoS One. 9:e918242014. View Article : Google Scholar : PubMed/NCBI

26 

Yuryev A: PCR primer design using statistical modeling. Methods Mol Biol. 402:93–104. 2007. View Article : Google Scholar : PubMed/NCBI

27 

Wangkumhang P, Chaichoompu K, Ngamphiw C, Ruangrit U, Chanprasert J, Assawamakin A and Tongsima S: WASP: A Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations. BMC Genomics. 8:2752007. View Article : Google Scholar : PubMed/NCBI

Related Articles

  • Abstract
  • View
  • Download
  • Twitter
Copy and paste a formatted citation
Spandidos Publications style
Peng J, Wei K, Zhao X, Yang K, Wang H, Zhang Y, Guo M, He J, Wu H, Li Y, Li Y, et al: Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS. Mol Med Rep 16: 2726-2732, 2017.
APA
Peng, J., Wei, K., Zhao, X., Yang, K., Wang, H., Zhang, Y. ... Fu, W. (2017). Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS. Molecular Medicine Reports, 16, 2726-2732. https://doi.org/10.3892/mmr.2017.6883
MLA
Peng, J., Wei, K., Zhao, X., Yang, K., Wang, H., Zhang, Y., Guo, M., He, J., Wu, H., Li, Y., Zhao, N., Huang, Q., Fu, W."Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS". Molecular Medicine Reports 16.3 (2017): 2726-2732.
Chicago
Peng, J., Wei, K., Zhao, X., Yang, K., Wang, H., Zhang, Y., Guo, M., He, J., Wu, H., Li, Y., Zhao, N., Huang, Q., Fu, W."Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS". Molecular Medicine Reports 16, no. 3 (2017): 2726-2732. https://doi.org/10.3892/mmr.2017.6883
Copy and paste a formatted citation
x
Spandidos Publications style
Peng J, Wei K, Zhao X, Yang K, Wang H, Zhang Y, Guo M, He J, Wu H, Li Y, Li Y, et al: Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS. Mol Med Rep 16: 2726-2732, 2017.
APA
Peng, J., Wei, K., Zhao, X., Yang, K., Wang, H., Zhang, Y. ... Fu, W. (2017). Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS. Molecular Medicine Reports, 16, 2726-2732. https://doi.org/10.3892/mmr.2017.6883
MLA
Peng, J., Wei, K., Zhao, X., Yang, K., Wang, H., Zhang, Y., Guo, M., He, J., Wu, H., Li, Y., Zhao, N., Huang, Q., Fu, W."Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS". Molecular Medicine Reports 16.3 (2017): 2726-2732.
Chicago
Peng, J., Wei, K., Zhao, X., Yang, K., Wang, H., Zhang, Y., Guo, M., He, J., Wu, H., Li, Y., Zhao, N., Huang, Q., Fu, W."Wild‑type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS". Molecular Medicine Reports 16, no. 3 (2017): 2726-2732. https://doi.org/10.3892/mmr.2017.6883
Follow us
  • Twitter
  • LinkedIn
  • Facebook
About
  • Spandidos Publications
  • Careers
  • Cookie Policy
  • Privacy Policy
How can we help?
  • Help
  • Live Chat
  • Contact
  • Email to our Support Team