Tested in:	Maize
Genetic target:	ADH1
Description:	Quantitative PCR method for detection of maize alcohol dehydrogenase 1 gene (ADH1) (EURL GMFF, 2005)
Comments:	Based on scientific evidence, the adh1 (70 bp) endogenous reference gene assay targets a region in the maize genome that shows a sequence polymorphism, which may affect the efficiency of amplification (Broothaerts et al., 2008). Users of this event-specific quantification method should, therefore, replace the maize adh1 (70 bp) gene assay with the hmg gene assay (or any other suitable maize reference gene assay). Bridging experiments by the EURL-GMFF [EU-RL GMFF validation report (13 November 2013): Report on the verification of the performance of 1507, 59122, MON 810 and NK603 event-specific PCR-based methods applied to DNA extracted from stack maize 1507 x 59122 x MON 810 x NK603 (http://biotech-staging.jrc.it/StatusOfDossiers.aspx)] have demonstrated that this event-specific quantification method performs adequately in combination with hmg as reference gene target. Taxon-specific methods may generate amplicons with different sequence or length in different plant varieties.

Specificity:	Species specific
Analysis type:	Quantitative
Assay:	Real-time PCR (Simplex)
Amplicon sequence:	CCTTCTTGGCGGCTTATCTGTCTTAGGGGCAGACTCCCGTGTTCCCTCGGATCTTTGGCCATGAGGCTGG Genbank ID: AY691949
Amplicon size:	70 bp
Oligos:

Type	Name(s)	Sequence	Length	Target
Primer forward	Adh1 primer F /	CCTTCTTGGCGGCTTATCTG	20 bp	ADH1
Primer reverse	Adh1 primer R /	CCAGCCTCATGGCCAAAG	18 bp	ADH1
Probe	Adh1 probe PR /	FAM-CTTAGGGGCAGACTCCCGTGTTCCCT-TAMRA	26 bp

QT-EVE-ZM-007 QT-EVE-ZM-009 QT-EVE-ZM-008