Analysis of the Copy Number of Herbicide Resistant Marker Gene EPSP in Transgenic Maize by Droplet Digital PCR

doi:10.7668/hbnxb.2017.03.011

Abstract

Abstract: The droplet digital PCR (ddPCR) method to evaluate the exogenous gene copy number in genetically modified crops (GM crops) as the example from genetically modified maize was developed,and some T₀ transgenic maize plants with single copy of EPSP gene were selected from all the samples to be detected.The results were also compared with those from quantitative real-time PCR (qRT-PCR),as could be seen from the experimental datas,the results from qRT-PCR(TaqMan) and ddPCR for EPSP were consistent,especially the single copy detection results of both in a high degree of consistency.And the ddPCR method was easy to operate,the result was repeatable and accurate.Therefore,the ddPCR would be well developed as a novel method for estimating transgenic copy number with high accuracy,which might be widely used in the exogenous genes copy number analysis in GM crops in the near future.

Key words: Droplet digital PCR, Genetically modified maize, Exogenous gene, Copy number, Absolute quantification

CLC Number:

S513.03

WANG Ben, ZHANG Chun, LI Xianglong, ZHANG Zhongbao, ZOU Huawen, WU Zhongyi. Analysis of the Copy Number of Herbicide Resistant Marker Gene EPSP in Transgenic Maize by Droplet Digital PCR[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(3): 70-76. doi: 10.7668/hbnxb.2017.03.011.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2017.03.011

http://www.hbnxb.net/EN/Y2017/V32/I3/70

References

[1] Heyries K A,Tropini C,Vaninsberghe M,et al.Megapixel digital PCR[J].Nature Methods,2011,8(8):649-651.
[2] 万艳,李丽玲,陈小佳.应用实时荧光定量PCR检测外源基因拷贝数的新方法[J].暨南大学学报:自然科学与医学版,2009,30(3):310-313.
[3] 王爱民.实时荧光定量PCR(TaqMan)法测定外源基因的拷贝数[J].广西植物,2009,29(3):408-412.
[4] Whale A S,Huggett J F,Cowen S,et al.Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation[J].Nucleic Acids Research,2012,40(11):e82.
[5] 姜羽,胡佳莹,杨立桃.利用微滴数字PCR分析转基因生物外源基因拷贝数[J].农业生物技术学报,2014,22(10):1298-1305.
[6] Sykes P J,Neoh S H,Brisco M J,et al.Quantitation of targets for PCR by use of limiting dilution[J].Bio Techniques,1992,13(3):444-449.
[7] Vogelstein B,Kinzler K W.Digital PCR[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(16):9236-9241.
[8] Abyzov A,Mariani J,Palejev D,et al.Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells[J].Nature,2012,492(7429):438-442.
[9] Boettger L M,Handsaker R E,Zody M C,et al.Structural haplotypes and recent evolution of the human 17q21.31 region[J].Nature Genetics,2012,44(8):881-885.
[10] Hindson B J,Ness K D,Masquelier D A,et al.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number[J].Analytical Chemistry,2011,83(22):8604-8610.
[11] Nair V D,Ge Yongchao,Balasubramaniyan N,et al.Involvement of histone demethylase LSD1 in short-time-scale gene expression changes during cell cycle progression in embryonic stem cells[J].Molecular and Cellular Biology,2012,32(23):4861-4876.
[12] Gorbachev A Y,Fisunov G Y,Izraelson M,et al.DNA repair in Mycoplasma gallisepticum[J].BMC Genomics,2013,14(1):726.
[13] Jiang Ke,Ren Chunyan,Nair V D.MicroRNA-137 represses Klf4 and Tbx3 during differentiation of mouse embryonic stem cells[J].Stem Cell Research,2013,11(3):1299-1313.
[14] Morisset D,tebih D,Milavec M,et al.Quantitative analysis of food and feed samples with droplet digital PCR[J].PLoS One,2013,8(5):e62583.
[15] Corbisier P,Bhat S,Partis L,et al.Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction[J].Analytical and Bioanalytical Chemistry,2010,396(6):2143-2150.
[16] Burns M J,Burrell A M,Foy C A.The applicability of digital PCR for the assessment of detection limits in GMO analysis[J].European Food Research and Technology,2010,231(3):353-362.
[17] Shlush L I,Zandi S,Mitchell A,et al.Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia[J].Nature,2014,506(7488):328.
[18] Johnson B E,Mazor T,Hong Chibo,et al.Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma[J].Science,2014,343(6167):189-193.
[19] Miyaoka Y,Chan A H,Judge L M,et al.Isolation of single-base genome-edited human iPS cells without antibiotic selection[J].Nature Methods,2014,11(3):291-293.
[20] Abdel-wahab O,Klimek V M,Gaskell A A,et al.Efficacy of intermittent combined RAF and MEK inhibition in a patient with concurrent BRAF-and NRAS-mutant malignancies[J].Cancer Discovery,2014,4(5):538-545.
[21] Rački N,Morisset D,Gutierrez-aguirre I,et al.One-step RT-droplet digital PCR:a breakthrough in the quantification of waterborne RNA viruses[J].Analytical and Bioanalytical Chemistry,2014,406(3):661-667.
[22] Rothrock M J,Hiett K L,Kiepper B H,et al.Quantification of zoonotic bacterial pathogens within commercial poultry pro-cessing water samples using droplet digital PCR[J].Advances in Applied Microbiology,2013,3(5):403-411.
[23] Zhao Hui,Wilkins K,Damon I K,et al.Specific qPCR assays for the detection of Orf virus,Pseudocowpox virus and Bovine papular stomatitis virus[J].Journal of Virological Methods,2013,194(1/2):229-234.
[24] Kelley K,Cosman A,Belgrader P,et al.Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay[J].Journal of Clinical Microbiology,2013,51(7):2033-2039.
[25] Laurie M T,Bertout J A,Taylor S D,et al.Simultaneous digital quantification and fluorescence-based size characterization of massively parallel sequencing libraries[J].BioTechniques,2013,55(2):61-67.
[26] White R A,Grassa C J,Suttle C A.Draft genome sequence of exiguobacterium pavilionensis strain RW-2,with wide thermal,salinity,and pH tolerance,isolated from modern freshwater microbialites[J].Genome Announcements,2013,1(4):e00513-e00597.
[27] Cangi M G,Biavasco R,Cavalli G,et al.BRAFV600E-mutation is invariably present and associated to oncogene-induced senescence in Erdheim-Chester disease[J].Annals of the Rheumatic Diseases,2015,74(8):1596-1602.
[28] Chong I Y,Cunningham D,Barber L J,et al.The genomic landscape of oesophagogastric junctional adenocarcinoma[J].The Journal of Pathology,2013,231(3):301-310.
[29] Frésard L,Leroux S,Servin B,et al.Transcriptome-wide investigation of genomic imprinting in chicken[J].Nucleic Acids Research,2014,42(6):3768-3782.
[30] Wang I X,Core L J,Kwak H,et al.RNA-DNA differences are generated in human cells within seconds after RNA exits polymerase Ⅱ[J].Cell Reports,2014,6(5):906-915.
[31] Chen Rui,Mias G I,Li-pook-than J,et al.Personal omics profiling reveals dynamic molecular and medical phenotypes[J].Cell,2012,148(6):1293-1307.
[32] Holmberg R C,Gindlesperger A,Stokes T,et al.Akonni TruTip(^®) and qiagen(^®) methods for extraction of fetal circulating DNA-evaluation by real-time and digital PCR[J].PLoS One,2013,8(8):e73068.
[33] Gu Wei,Koh W,Blumenfeld Y J,et al.Noninvasive prenatal diagnosis in a fetus at risk for methylmalonic acidemia[J].Genetics in Medicine,2014,16(7):564-567.
[34] Pornprasert S,Prasing W.Detection of alpha(0)-thalassemia South-East Asian-type deletion by droplet digital PCR[J].European Journal of Haematology,2014,92(3):244-248.
[35] Fu Wei,Zhu Pengyu,Wang C,et al.A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment[J].Scientific Reports,2015,5(1):12715.
[36] Sedlak R H,Jerome K R.Viral diagnostics in the era of digital polymerase chain reaction[J].Diagnostic Microbiology and Infectious Disease,2013,75(1):1-4.
[37] 李志亮,吴忠义,杨清,等.花粉管通道法在玉米基因工程改良中的应用[J].玉米科学,2010,18(4):71-73,76.
[38] 王艳杰,申家恒.花粉管通道法转基因技术的细胞胚胎学机理探讨[J].西北植物学报,2006,26(3):628-634.
[39] 王秀君,郎志宏,陆伟,等.利用花粉管通道法将耐草甘膦基因mG2-epsps导入玉米自交系的研究初报[J].中国农业科技导报,2008,10(4):56-62.
[40] 王永锋,栾雨时,高晓蓉.花粉管通道法在植物转基因中的研究与应用[J].东北农业大学学报,2004,35(6):764-768.
[41] 陈向明.用CTAB法提取植物DNA的技术改进[J].合肥教育学院学报,2000(4):14-16.
[42] Zhang Dabing,Guo Jinchao.The development and standardization of testing methods for genetically modified organisms and their derived products[J].Journal of Integrative Plant Biology,2011,53(7):539-551.
[43] Vaucheret H,Béclin C,Elmayan T,et al.Transgene-induced gene silencing in plants[J].The Plant Journal,1998,16(6):651-659.
[44] Hayden R T,Gu Z,Ingersoll J,et al.Comparison of droplet digital PCR to Real-time PCR for quantitative detection of cytomegalovirus[J].Journal of Clinical Microbiology,2013,51(2):540-546.

Please choose a citation manager

Content to export