Standard addition quantitative real-time PCR (SAQPCR): a novel approach for determination of transgene copy number avoiding PCR efficiency estimation.

ABSTRACT: Quantitative real-time polymerase chain reaction (qPCR) has been previously applied to estimate transgene copy number in transgenic plants. However, the results can be erroneous owing to inaccurate estimation of PCR efficiency. Here, a novel qPCR approach, named standard addition qPCR (SAQPCR), was devised to accurately determine transgene copy number without the necessity of obtaining PCR efficiency data. The procedures and the mathematical basis for the approach are described. A recombinant plasmid harboring both the internal reference gene and the integrated target gene was constructed to serve as the standard DNA. It was found that addition of suitable amounts of standard DNA to test samples did not affect PCR efficiency, and the guidance for selection of suitable cycle numbers for ana

SUBMITTER: Huang Y 

PROVIDER: S-EPMC3538589 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature