Pakistan Science Abstracts
Article details & metrics
No Detail Found!!
Tandem Recombinant Plasmid Construction as Positive Control for Pik3ca H1047r Detection Based on SYBR Green I QPCR.
Author(s):
1. Bugi Ratno Budiarto: Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong, Bogor 16911, West Java, Indonesia
2. I Made Artika: Department of Biochemistry, Bogor Agricultural University. Jl. Raya Darmaga, Kampus IPB, Bogor 16680, West Java, Indonesia
3. Azamris: Division of Surgical Oncology Medical School of Andalas University-M.Djamil Hospital, Jl Perintis Kemerdekaan No. 94, Padang. Indonesia
4. Nahdaturrugaisiyah: Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong, Bogor 16911, West Java, Indonesia: Department of Biochemistry, Bogor Agricultural University. Jl. Raya Darmaga, Kampus IPB, Bogor 16680, West Java, Indonesia
5. Desriani: Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong, Bogor 16911, West Java, Indonesia
Abstract:
PIK3CA H1047R mutation is found in breast cancer in high frequency and its detection could be applied as prognosis and predictive factor for trastuzumab therapy. qPCR is one of the simplest and robust method for PIK3CA H1047R detection. Provision of positive control for PIK3CA H1047R detection based on qPCR will support data analysis efficiently and avoid false negative result. In this research, we constructed a tandem recombinant plasmid (pGEM-tandPIK3CA) as positive control for Tm Shift SYBR Green I qPCR-based of PIK3CA H1047R detection system by ligating wild-type and PIK3CA H1047R fragments tandemly into pGEM-T Easy. The tandem plasmid was confirmed by restriction, DNA sequencing and qPCR. As a result, pGEMtandPIK3CA has been successfully constructed and confirmed. Statistical analysis shows high repeatability and reproducibility with % CV of <25%. The main advantage of this tandem positive control is its ability to serve as positive control for both wild-type PIK3CA and PIK3CA H1047R simultaneously, therefore improving the efficiency of the detection system.
Page(s): 735-742
DOI: DOI not available
Published: Journal: Pakistan Journal of Biotechnology, Volume: 15, Issue: 3, Year: 2018
Keywords:
Keywords are not available for this article.
References:
[1] Muhartono , Ermin, R.,P.D.J. Bayu, 2018.Mucoxin enhanced transcription and protein expression of p53 in breast cancer cell line T47D,Pak. J. Biotechnol 15 219 -224
[2] Bhargava , R.,Sahoo , S.,Esposito , N.N.,B.Chen, 2011.Pathology of breast carcinoma: diagnostic, prognostic and therapeutic issues and challenges, 1 2 -
[3] Susianti , S.,Yanwirasti , Y.,Darwin , E.,J.Jamsari, 2018.The cytotoxic effects of purple nutsedge (Cyperus rotundus L.) tuber essential oil on the HeLa cervical cancer cell line,Pak. J. Biotechnol 15 77 -81
[4] Miller , T.W., 2012.Initiating breast cancer by PIK3CA mutation,Breast Cancer Res 14 301 -302
[5] Ang , D.,O'Gara,R. , Schilling,A. , Beadling,C. , Warrick,A. , Troxell,M.L., 2013.Novel method for PIK3CA mutation analysis locked nucleic acid-PCR sequencing,J. Mol. Diagn 15 312 -18
[6] Li , W.M.,Hu,Zhou , L.L.,Feng , Y.M.,Wang , Y.Y.,J.Fang, 2016.Highly sensitive detection of the PIK3CAH1047R mutation in colorectal cancer using a novel PCR-RFLP method,BMC Cancer 16 454 -464
[7] Gkeka , P.,Evangelidis , T.,Pavlaki , M.,Lazani , V.,Christoforidis , S.,Agianian , B., 2014.Investigating the structure and dynamics of the PIK3CA wild-type and H1047R oncogenic mutant,PLoS Comput. Biol 10 1 -12
[8] Engelman , J.A.,Chen , L.,Tan , X.,Crosby , K.,Guimaraes , A.R.,Upadhyay , R., 2008.Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancer,Nat. Med 14 1351 -1356
[9] Higgins , M.J.,Jelovac , D.,Barnathan , E.,Blair , B.,Slater , S.,Powers , P., 2012.Detection of tumor PIK3CA status in metastatic breast cancer using peripheral blood,Clin. Cancer Res 18 3462 -3469
[10] Stemke-Hale , K.,Gonzalez-Angulo , A.M.,Lluch , A., 2008.An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer,Cancer Res 68 6084 -6091
[11] Karakas , B.,Bachman , K.E.,B.H.Park, 2006.Mutation of the PIK3CA oncogene in human cancers,Br. J. Cancer 94 455 -59
[12] Berns , K.,Horlings , H.M.,Hennessy , B.T.,Madiredjo , M.,Hijmans , E.M.,Beelen , K., 2007.A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer,Cancer Cell 12 395 -402
[13] Alvarez-Garcia , V.,Bartos , C.,Keraite , I.,Trivedi , U.,Brennan , P.M.,Kersaudy-Kerhoas , M., 2018.A simple and robust real-time qPCR method for the detection of PIK3CA mutations,Nature 8 4290 -4299
[14] Niwa , H.,Anzai , T. and S.,Hobo, 2007.Construction of a recombinant plasmid as reaction control in routine PCR for detection of contagious equine metritis (CEM-PCR),J. Vet. Med. Sci 69 1199 -1201
[15] Hoorfar , J.,Malorny , B.,Abdulmawjood , A.,Cook , N.,Wagner , M.,P.Fach, 2004.Practical considerations in design of internal amplification controls for diagnostic PCR assays,J. Clin. Microbiol 42 1863 -1868
[16] Majidzadeh , K.,Mohseni , A.,M.Soleimani, 2014.Construction and evaluation of a novel internal positive control (IPC) for detection of Coxiella burnetii by PCR,Jundishapur J. Microbiol 7 -
[17] Broeders , S.,Huber , I.,Grohmann , L.,Berben , G.,Taverniers , I.,Mazzara , M., 2014.Guidelines for validation of qualitative real-time PCR methods,Trends Food Sci. Technol 37 115 -126
[18] Hou , L.,Zhang , X.,Li , Y.,Chen , S.,Qu , H.,Yu , J., 2016.Rapid screening of recombinant plasmids by direct colony quantitative real-time PCR,Advances in Bioscience and Biotechnology 7 428 -433
[19] Morlan , J.,Baker , J. and D.,Sinicropi, 2009.Mutation detection by real-time PCR: a simple, robust and highly selective method,PLoS ONE 4 -
[20] Wang , T.,Wang , D.,Lyu , Y.,Feng , E.,Zhu , L.,Liu , C., 2018.Construction of a high-efficiency cloning system using the Golden Gate method and I-SceI endonuclease for targeted gene replacement in Bacillus anthracis,J. Biotechnol 271 8 -16
Citations
Citations are not available for this document.
0

Citations

0

Downloads

4

Views