Abstract:
In transgenic plants, transgene copy number can
greatly influence the expression level and genetic
stability of the target gene. Thus, making estimation of
transgene copy number and determination of the
expression levels an important area of genetically
modified crop research.
The analysis of transgenic plants can be as
important as the transformation process itself. After
producing the primary transformants, it is very
important to decide which plants contain the transgene
and in how many copies[7]. While multiple copies are
useful for over-expression experiments, single or low
copy transformation events are preferred for most
applications because they are stable over several
generation of subsequent breeding[24].
The analysis of transgene integration has been
recently relied on PCR-based methods, which require
only small amounts of plant material and are easily
automated for high-throughput quantita tion .
Conventional PCR is commonly used for screening
large numbers of putative transformants for transgenic
sequences[23] and as an alternative to marker selection
for segregation analysis[18]. However, conventional PCR
analysis is plagued by false positives from
contamination because a band with the size of the
desired product is counted as signal, regardless of the
intensity. Transgene copy number has traditionally been
estimated by Southern analysis, although recently other
methods, including comparative genomic hybridization,
fluorescence in situ hybridization, multiplex amplifiable
probe hybridization and microarray have been applied
to copy number estimation. Unfortunately, all of these
methods are laborious and time-consuming, require
considerable amount of DNA from fresh or frozen
samples and often involve the use of hazardous
radioisotopes[34].
Real time PCR (ABI Prism 7700 Sequence
Detection System) has been recently established as a
rapid and sensitive technique for precise quantitation[13].
With real-time PCR as a quantification tool, it is
possible to rapidly analyze large numbers of putative
transformants from high-throughput transformation
procedures[7]. Real-time PCR was initially used in plant
research as a highly specific and sensitive detection
