510 Chapter 29
these junctions is present in the genome of
the di - or polyploid transformant, as com-
mercialized GMO lines are generally hetero-
zygous (Berdal and Holst - Jensen 2001 ).Relative and Absolute Quantifi cation
Absolute quantifi cation is the determination
of the amount or copy number of the target
DNA sequence in the analyzed sample, while
relative quantifi cation is the determination of
GMO percentage referred to the endogenous
reference gene. Relative quantifi cation is
achieved by the ratio of two quantifi cation
values: the GMO - specifi c gene versus the
endogenous reference control gene. The ratio
is expressed in percentage of genome/genome
(g/g%) or of weight/weight (w/w%). A criti-
cal point for the relative quantifi cation is that
PCR effi ciencies of each PCR system must
be comparable. Another analytical strategy is
the use of the 2^ −^ Δ^ Δ CT analysis (Livak andCommonly used techniques for plant
transformation introduce a randomized and
unknown copy number of the construct into
the host plant, and it may be diffi cult to
accurately determine the copy number of the
integrated sequence as well as the location
in the nuclear genome. If the detection
method targets a certain part of the gene con-
struct, exact absolute quantifi cation becomes
uncertain if the inserted copy number is not
known. Consequently, the ideal DNA target
sequence for GMO quantifi cation is a
sequence found only once in a stable and
known copy number per genome. For this
reason, several works describe the character-
ization of junction regions between the
host plant genome and the transgene
(Zimmermann et al. 2000 ; Holck et al. 2002 ;
Hernandez et al. 2003a ; Windels et al.
2003 ; Nielsen et al. 2004 ; Taverniers et al.
2004 ) that are unique for a single transforma-
tion event (event - specifi c). Only one copy of
Table 29.3. PCR methods for analysis of endogenous reference genes. c PCR : conventional PCR.
RT i - PCR : real - time PCR. Modifi ed from Hernandez et al. (2005)
Plant species Endogenous reference genes Technique Reference
Eggplant β - fructosidase RTi - PCR Chaouchi et al., 2008
Maize Zeine cPCR Studer et al., 1997
Invertase ( ivr1 ) cPCR Ehlers et al., 1997
Zeine RTi - PCR Va ï tilingom et al., 1999
High mobility group protein ( hmg ) cPCR Zimmermann et al., 1998a
Adh1, hmga, ivr1, zeine RTi - PCR Hernandez et al., 2004b
Pepper β - fructosidase RTi - PCR Chaouchi et al., 2008
Potato Sucrose - synthase cPCR Akiyama et al., 2002
Metallo - carboxypeptidase ( pci ) RTi - PCR Hernandez et al., 2003b
β - fructosidase RTi - PCR Chaouchi et al., 2008
Rapeseed Acetyl CoA carboxylase ( acc1 ) RTi - PCR Hernandez et al., 2001
Acetyl CoA carboxylase ( acc1 ) RTi - PCR Schmidt and Rott, 2006
Rice Sucrose phosphate synthase RTi - PCR Ding et al., 2004
Soybean Lectin ( le1) cPCR Meyer et al., 1996
Heat - shock protein ( HSP ) cPCR Krech, 1997
Lectin ( le1) cPCR Wurz et al., 1998
Tomato Metallo - carboxypeptidase ( mpci ) RTi - PCR Hernandez et al., 2003b
LAT52 RTi - PCR Yang et al., 2005
β - fructosidase RTi - PCR Chaouchi et al., 2008
Wheat 25S – 18S ARNr cPCR Allmann et al., 1993
Low Molecular Weight glutenin RTi - PCR Terzi et al., 2003
Waxy - D1 RTi - PCR Ida et al., 2005