Post on 19-Feb-2022
Experimental basic steps
DNA residues purification
Qalitative PCR amplification at 15 SSRs loci
Allele sizing by MEGABace 500 (GE Healthcare)
NERETO GRANOIR
ABROSTINE SAN COLOMBANO
ABRUSCO VERMENTINO BIANCO
ANSONICA MAZZESE
B45BB GIACCHE'
BARSAGLINA ALEATICO
BIV1-420 MONTEPULCIANO D'ABRUZZO
BRONTOLO NEGROTINO
C2 GRAND NOIR
CANAIOLO NERO PETIT VERDOT
CILIEGIOLO MERLOT
FOGLIA FRASTAGLIATA GAMAY
FOGLIA TONDA GRANE’
M235 SYRAH
M238 CABERNET FRANC
MAMMOLO CABERNET SAUVIGNON
POLLERA TEROLDEGO
PRUGNOLO GENTILE ANCELLOTTA
PUGNITELLO TINTURIER
SANGIOVESE APSG2 REFOSCO DAL PEDUNCOLO ROSSO
SANGIOVESE R24 MORONE
SANGOVESE F9 COLORINO
VERDELLO BIOT.PITIGL. RASPO ROSSO
VERDELLO VERMENTINO NERO
WINE TRACK AUSTRALIA
BRL HARDY COMPANY
BOTTLE WITH SEAL IN WHICH THERE IS A CODE WHERE DNA EXTRACTED FROM GRAPEVINE IS MIXED WITH INORGANIC CHEMICALS AND USED AS AUTHENTICITY LABEL MATRIX TM
AND THE 'COMBINED WITH INK, MARCATORE, RESINS ETC.
LABELS ARE READ BY AN AN ELECTRONIC SCANNER THAT ENSURES TRACEABILITY OF BOTTLES
WINE TRACK
EFSA (European Food Safety Authority) STABLE ISOTOPE ANALYSIS (C,H,O)
ISOTOPE RELATIVE RATIO DEPENDS ON GEOGRAPHICAL PROVENIENCE, CLIMATE CONDITIONS
Chemometric anthocyanines wine profiling (Iztok Joz et al. Analytica Chimica ActaVolume 513, 18 June 2004, Pages 277-282 ) • Merlot, Cabernet sauvignon, Pinot noir • Environment effects
DNA and consumer confidence (EMBO reports 4, 3, 232ミ234 (2003)) In forensics DNA testing is fundamental for individual identification PCR is the key technique, which allows to analyse even DNA highly degraded (fossil specimens) or present in traces (a few or even one hairbulb, biological specimens present in crime scenes)
• Identify potential suspects whose DNA may match evidence left at crime scenes • Identify crime and catastrophe victims • Establish paternity and other family relationships
IN MEDICINA FORENSE SI ESTRAE DNA DA CELLULE RESIDUE, OSSA,TESSUTI,CAPELLI, RESIDUI DI LIQUIDI ORGANICI E SANGUE ECC.
DNA purification from WINE
DNA purification by use of linear acrylamide (Dràbek et al., Eur Food Res Technol 2008) or other chelating agents (Argylla technologies, Tucson, AZ, USA)
Siliceous matrix-based kits (Qiagen)
Chemical extraction (SDS, CTAB) after an initial massive precipitation step (Siret et al., J. Agric. Food Chem. 2002; Savazzini et al. Analytica Chimica Acta 2006)
• Siret et al, 2000
Centrifugazione
sovranatante Pellet
2 X CTAB
2 X clorof.-isomilico +ppt in alcool e sale
DNA crudo
DNA crudo
Proteinasi K
1 x Estrazione fenol-clorof.
Ppt 2 vol alcool + sale
Purificazione mediante colonne
Qiagen
2° prelievo
1° eluizione
2° eluizione
• Left: DNA fractions extracted from wine (Wang, personal communication). Right: DNA extracted following a modification of the Siret protocol (Siret et al.,2000). DNA presence is evidenced with coloured ovals. Lambda HindIII is used as molecular weight standard (asterisk)
• Residual grapevine DNA is estimated to be 2 picograms-4000picograms/ml
* *
Sangiovese and more?..
SANGIOVESE genotype wine 179-185 179-185-189
175 175 175 175
159-161 159-161 135 135-137
260-262 260-262-254-256 156-162 156-162-166
240 240
Quantitative assay by Real time-PCR
A new set of three- and tetra- nucleotidic markers under development (in collaboration with VIGNA project)
Apply molecular genotyping to the vineyards to establish a detailed program of analytical traceability for each producer (from field to bottle.. to meet real genetic
diversity population)