Document Detail


Estimating the copy number of transgenes in transformed cotton by real-time quantitative PCR.
MedLine Citation:
PMID:  23143487     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
Transgenic cotton has widely been employed both in commercial cultivation and basic research. It is essential to determine which plants contain the transgene and in how many copies after transgenic cotton plants are generated. A TaqMan quantitative real-time polymerase chain reaction (Tq RT-PCR) method is described here to examine transgene copy number in transgenic cotton plants. The estimation of two transgene elements, the target gene of green fluorescence protein (GFP) and the selective gene of neomycin phosphotransferase II (NPTII), is used as an example to detail each step in Tq RT-PCR procedure, including endogenous reference gene selection, reference plasmid construction, primer-probe design, DNA extraction, real-time PCR, and data analysis. Comparing with traditional approach-Southern hybridization -analysis, this method can be used efficiently in screening large number of T0 transgenic cotton plants at early stage of transformation process as well as identifying transgene homozygotes in a segregation population.
Authors:
Chengxin Yi; Yan Hong
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  958     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2013  
Date Detail:
Created Date:  2012-11-12     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  109-30     Citation Subset:  IM    
Affiliation:
JOil (S) Pte Ltd, 1 Research Link, National University of Singapore, Singapore, Singapore.
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