hydrogen bonds (Memelink et al. 1994; Sambrook and Russell 2001). The wells are washed
and incubated with an enzyme-linked conjugate that will produce a color change when
flooded with substrate. The degree of color change can be judged by the naked eye or quan-
tified on a spectrophotometer (Fig. 11.2). Semiquantitative ELISAs may also be used to
estimate transgenic protein production.
11.4 Definitive Molecular Characterization
11.4.1 Intact Transgene Integration
Copy number, or the number of times a transgene is inserted into the plant genome, is
demonstrated by Southern blot analysis. Southern blot analysis is a multistep process that
takes several days. It entails isolating sufficient (tens of micrograms) quantities of
genomic DNA per plant, digesting the DNA to completion using a carefully chosen restric-
tion enzyme or enzymes, and separating the fragments on a gel according to size. The DNA
is transferred to a membrane (blot), and then the immobilized genomic DNA is hybridized
with a radiolabeled probe (Feinberg and Vogelstein 1983, 1984) or a nonisotopically
labeled probe (Langer et al. 1981) (Fig. 11.3).
Southern blots require high-quality high molecular weight genomic DNA (e.g.,20 kb).
Extraction of high molecular weight DNA is critical; if the DNA is degraded or sheared, the
bands on a Southern blot will not be distinct, since there will be smearing of DNA that is
hybridized with probe. If the DNA is uncut, it will produce an unacceptable high molecular
weight artifact after hybridization (Birch 1997; Potrykus 1991). After the DNA is digested
to completion, the DNA fragments are separated on an agarose gel (Fig. 11.4a). DNA has a
negative charge because of the phosphate groups and will travel toward the anode, which
has a positive charge. Once the fragments have separated, the gel is placed on a nylon mem-
brane (blot), under added weight and absorbent towels, and the fragments migrate into the
blot by capillary action (Sambrook and Russell 2001). This process is termed “blotting,”
and the nylon filter is easier to handle in subsequent applications than the agarose gel,
and is also more conducive to probing. In the traditional Southern blot, the membrane is
Figure 11.3.The process of DNA gel blot analysis (Southern blotting and hybridization): (a)
digested DNA fragments are separated according to size on an agarose gel; (b) DNA fragments are
transferred to a blot; (c) autoradiograph of the blot after hybridization with radiolabeled, single-
stranded complementary DNA probe.
280 TRANSGENIC PLANT ANALYSIS