â¢Invented by Kary B. Mullis in 1983 (won Nobel prize in 1993); and it uses primers to exponentially amplify a specific DNA. Requirements for the PCR reaction.
Geoffrey Muriira, Ph.D Mol. Bio Lab KEBS
Why and Who needs GMO measurements? Those who want GM varieties: trait purity testing such as Biotech producers, seed co., farmers Variety conversion Track different products Assure compliance of biotech varieties to commercial purity standards b) Those who don’t: adventitious presence testing; seed co. farmers, food and feed industry, enforcement authorities, testing labs;
a)
Monitoring after commercial release Assure product segregation Compliance with legislation (International, and national standards)
Potential Benefits of GMOs Producer Benefits Increased yields (by preventing crops against diseases, pests) Decreased pesticides, herbicides and fungicides Decreased costs Improving Nutrition value
Potential Concerns Human health (toxicity, allergenicity,
altering microflora…) Environmental risks (transgene escape, harm to non-target organisms, pest resistance and super weeds) Antibiotic resistance
ISO standards on GMO Measurements Methods of analysis for the detection of
genetically modified organisms and derived products ISO 24276- General requirements and definitions
ISO 21571- Nucleic acid extraction 21569- Qualitative nucleic acid based methods 21570- Quantitative nucleic acid based methods 21572- Protein based methods ISO 21568- Sampling (Not yet finalized)
Procedures in the lab Sampling
Laboratory Sample Nucleic Acid Extraction Qualitative PCR Protein based methods Screening method If required
Quantitative PCR
Construct/ Event specific method Identification
What genes are transferred? 1. Control sequences: promoters, terminators &
Introns 2. Genes for selectable markers i. Tolerance to antibiotics (amp, nptII) ii. Tolerance to herbicides (bar, pat) 3. Genes for new traits a) Enzymes; i. ii.
Tolerance to herbicides; EPSPS, bar, pat, gox New metabolic pathways (oils and fats, vitamins)
4. Other proteins e.g. insecticidal; Bt endotoxins, viral
coat proteins and lecithins 5. Anti-sense constructs e.g. Antipolygalaturonase and altered ethylene metabolism
The genetically modified glyphosate resistant Soya GTS 40-3-2 (Roundup Ready®17) Promoter
CaMV 35S
Coding region
CTP chloroplast transfer peptide sequence from Petunia hybrida for transition of EPSPS into the chloroplasts
EPSPS Enolpyruvyl shikimi-3-phosphate Synthase from the Agrobacterium
Terminator
T-NOS
PCR method for GMO detection Invented by Kary B. Mullis in 1983 (won Nobel
prize in 1993); and it uses primers to exponentially amplify a specific DNA Requirements for the PCR reaction DNA template (extracted from GM products) Primers DNA polymerase (Taq polymerase) dNTPs Buffers.
THE INCREASE IN PRODUCT IS EXPONENTIAL
Real Time PCR; Qualitative and Quantitative Measurements
Calibration for Maize Standard and P35S Log 10 Copy Numbers
5 Maize STD y = -0.2393x + 11.572 R² = 0.9951
4 3
P35s y = -0.2967x + 12.545 R² = 0.9993
2 1 0 0
10
20
30 Mean CT
40
50
Qualitative results P35S results
TNOS Results
Negative 42% (18)
Negative 60% (26)
TNOS Negative
Positive 58% (25)
Positive 40% (17)
TNOS Positive
0
0
10 20 30
Quantitative results
We quantified P35S in 5 samples
S/No 1 2 3 4 Bt 63 Rice
% GM content 40.78 43.63 39.00 44.31 1.23%
10
20 30
Labeling Legislations Kenya 1. 0 % threshold Japan 5.0 % threshold except refined products European Union 0.9% threshold, seeds 0% and non-authorized products 0.5% Most stringent (labeling even if not detectable i. e oils) Traceability USA No limit South Korea 3.0 % threshold and refined products exempted Australia/New Zealand 1% and refined products exempted Implications Traceability, construct technologies and consumer information Safety?
Thank You
for your attention
Balance of Risk and Benefit ?Would the world be better without penicillin
Penicillin-induced anaphylaxis causes up to deaths every year in the US, in Africa??? 1,000 ??Kenya people may have died due to 100,000 > allergic reactions to penicillin If penicillin were discovered today, it would fail all clinical trials & regulatory approval ..BUT
Penicillin has saved up to 100,000,000 lives Many of you owe your very existence to this drug
