marker (SM) probe (Fig. 11.7). Copy number can be determined relative to either T-DNA
border, but right border analysis is the best determinant of whether the T-DNA has
transferred to the plant genome because it tends to transfer more faithfully than does
the left border (Caplan et al. 1985). However, the nature of the markers in the genetic
construct and their usefulness as probes will ultimately determine which border is
appropriate to analyze.
- Copy number can be determined by using an enzyme that cuts once in the introduced
DNA, and then the genomic blot is probed with sequences on either side of the restriction
site. As an example, Sac1 cuts once in the introduced DNA, and the blot could be
probed with either the selectable marker (SM) or the gene of interest (GOI) (Fig. 11.7).
This method has been used in Biolistics, where multiple copies often integrate into
one chromosomal location (Taylor and Fauquet 2002). See Jordan (2000) for an
additional example.
Both means of determining copy number yield the same information. Band sizes will be
different from the plasmid control if the introduced DNA has integrated into the genome.
The researcher needs only to count the number of bands to determine the number of
times the DNA was integrated into the plant genome. If possible, independent transformants
produced using the same plasmid should be shown on a single blot, in addition to wild-type
control and plasmid DNA samples, to lessen the likelihood of misinterpreting data from a
single lane (Birch 1997). Using the same kind of analysis with T 1 plants or more advanced
generations, we can also determine which copies of the transgene are inherited in individual
progeny plants.
Real-time PCR (Higuchi et al. 1992, 1993) is a relatively new, high-throughput
procedure for determining transgene copy number in plants (Bubner and Baldwin 2004;
Ingham et al. 2001; Mason et al. 2002), but it should be used as a supplement to
Southern analysis (Bubner and Baldwin 2004). It is an automated PCR procedure that
monitors amplification of a DNA sequence in a microcentrifuge tube during repetitive
PCR cycles by use of a fluorescent dye that is bound to primer or the PCR product
itself whose signal increases in direct proportion to the PCR product (Reece 2004).
Bubner and Baldwin (2004) present an in-depth discussion of this method, including its
applications and limitations.
Figure 11.7.Integration of T-DNA in plant genome. The T-DNA from LB to RB is integrated in the
plant genome. Left border analysis would involve digestion withEcoR1 that cuts in the selectable
marker (SM) and into the plant genome and probing with SM cDNA or PCR fragment. AHindIII
digest could be used as a diagnostic Southern blot to test whether the entire promoter: coding
region: terminator of the gene of interest (GOI) was transferred in its entirety and is the
correct size. (LB¼left border; RB¼right border; Pro:SM:Term¼Promoter:Selectable
Marker:Terminator; Pro:GOI:Term:Promoter:Gene of Interest:Terminator.)
11.4. DEFINITIVE MOLECULAR CHARACTERIZATION 283