Detection methods of genetically modified organisms

1
Detection methods of genetically modified
organisms

• Swathi. R. Pullagurla
• Literature seminar
• May 10th 2010

2
Overview

• Goal
• Genetically modified organisms(GMOs)
• Detection methods of GMOs
• CE and Electrochemiluminescence
• Fluorescence Cross-correlation Spectroscopy
• Results
• Critiques
• Acknowledgements

3
Goal
To detect the target DNA in genetically
modified organisms using PCR and PCR free
techniques

• .

4
Genetically modified organisms

• Organisms with modified genome
• Exogenous gene
• Confers new characteristics

http//www.glofish.com, http//othersideoftheplan
et.com E. Mariotti, M. Minunni and M. Mascini,
Anal. Chim. Acta 453 (2002), pp.165-172
5
GMOs

• 81.3 million hectares of worlds area
• 60 of foods in US supermarkets contain GMOs
• European Union regulations stipulate the labeling
  of GM products with more than 0.9

• .

Beachy, R.N.(1999) Facing fear of
biotechnology. Science 285,335 Deisingh et al.
Food Research International, Vol 38, Issue 6,
July 2005, Pages 639-649
6
Significance of GMOS

• Increased global food production
• Increased resistance to pests and diseases
• Adaptability to harsh growing conditions
• Desirable functional and nutritional
  characteristics
• Tolerance to safe herbicides

http//biotech.cas.psu.edu
7
Roundup Ready Soy (RRS)

• Glyphosate-resistant crop
• developed by Monsanto (EPA 2002)
• Common elements in GMO constructs
• 35 S promoter from Cauliflower
• mosaic virus (p35S)
• Terminator from the nopaline
• synthetase gene of Agrobacterium
  tumefaciens (tNOS)

Farid. E. Ahmed, Trends in Biotechnology Vol.20
No.5 may 2002
8
GMO testing of food
Food sample
DNA Extraction
GMO screening 35S promoter PCR test
Positive
Negative
PCR test approved for GMOs
No GMO labeling
Negative (No authorized food)
Positive (GMO labeling)
9
Detection methods
Protein based testing methods

• Western Blot
• ELISA
• Lateral flow strip
• Southern blot
• PCR
• Quantitative real time PCR

DNA based testing methods
Biosensors Microarrays
Farid. E. Ahmed Trends in Biotechnology 20 (2002)
215-223
10
Why DNA based methods?

• Genetic code is universal and DNA is ubiquitous
  molecule
• Nucleic acids are thermo-stable whereas
  proteins are thermo-sensitive
• Low amounts of DNA can be amplified using PCR
  techniques
• Food proteins are not detectable upon processing

• Versatile
• Sensitive
• Specific
• Precise

E. Gachet et al./Trends in Food Science
Technology 9 (1999) 380388 http//www.chemistry.n
msu.edu/research/MARC/images/dna_strand.jpg
11

• Capillary Electrophoresis with
  Electrochemiluminiscent Detection for Highly
  Sensitive Assay of Genetically Modified Organisms
• Longhua Guo, Huanghao Yang, Bin Qui, Xueyang
  Xiao, Linlin Xue, Donghwan Kim and Guonan Chen
• Anal. Chem. 2009, 81, 9578-9584

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Objective
To investigate the conditions for DNA
amplification, CE-ECL separation and detection
13
Electrochemiluminescence

• Reactive species are generated from stable
  precursors at the surface of an electrode
• Ru(bpy)32 was the first inorganic complex to
  show ECL
• Annihilation mechanism is the general one for ECL
  generation

http//chemistry.illinois.edu/research/materials/s
eminar_abstracts
14
CE-ECL detection interface
1.Electrophoretic buffer reservoir 2.Coreactant
Injection entrance 3.ECL detection window 4. Pt
grounding electrode 5. Waste exit 6. Silver wire
quasi-reference electrode 7. Working electrode 8.
Pt Counter electrode 9. Separation capillary 10.
Detection capillary 11. Etched capillary porous
joint
15
Approach for GMO detection
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Polymerase Chain Reaction (PCR)

• Consists of 25-45 cycles
• Each cycle consists of 3 steps
• Denaturation of DNA (template) at 91-97oC
• Annealing of primers to the template
• 5oC below the lowest Tm of primers
• Extension of primers and attachment of dNTPs at
  optimum 72oC (100 bases/s)
• C N (En ) 2n
• C Copy number of target after PCR
• N Starting copy number
• En Amplification efficiency of cycle
• n Cycle number

http//users.ugent.be/avierstr/principles/pcrstep
s.gif
17
Phases in PCR
Traditional PCR detection
Area of detection for Real-time
http//www.savethefrogs.com/chytrid/images/ABI-rea
ltime-vs-conventional-PCR.pdf

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Real-Time PCR

• Measurement of gene expression levels
• Low DNA copy number detection
• Bacteria and virus detection
• Real-Time PCR chemistry
• SYBR Green (Molecular Probes)
• A fluorogenic dye that exhibits little
  fluorescence when in solution, but emits a strong
  fluorescent signal upon binding to
  double-stranded DNA

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Experimental procedure
20
Primer labeling
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Primers used in PCR
Primer Target Sequence Amplicon length (bp)
U-35S D-35S 35S CCGACAGTGGTCCCAAAGATG AGAGGAAGGGTCTTGCGAAGG 158
U-NOS D-NOS NOS GAATCCTGTTGCCGGTCTTG GCGGGACTCTAATCATAAAAACC 125
U-epsps D-epsps cp4-epsps GCAAATCCTCTGGCCTTTCC CTTGCCCGTATTGATGACGTC 145
U-lectin D-lectin lectin GGGTGAGGATAGGGTTCTCTG GCGATCGAGTAGTGAGAGTCG 210
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Optimized PCR conditions
Primer concentration Template DNA concentration Mg2 conc. dNTP concentration Anneal temp.
UD-lectin 0.1 µM UD- 35S 0.3 µM UD-NOS 0.35 µM UD - epsps- 0.5 µM 100 ng (per 25 µL) 1.5 mM 0.5 mM 59 C
23
Optimized CE conditions
Sieving matrix Buffer pH Separation voltage Injection time
PVP 2.5 Tris HCl- 20 mmol/L 8 300 V/cm 12 s
24

• Results

25
RT-PCR detection of RRS
35S labeled with Ru(phen)32
26
Stability of primerslabeled with Ru(phen)3 2
Time after labeling (day) Migration time (s) ECL count
0 834 10 8367 103
1 829 9 8218 136
2 833 11 8257 122
3 835 8 8244 146
4 822 12 8167 149
5 828 11 8259 131
6 837 10 8217 124
7 831 13 8266 157
U-lectin was tested
27
Certified reference material (CRM) standards of
RRS
Lectin
NOS
cp4-epsps
35S
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Reproducibility
Objective gene Size (bp) Average (count) RSD Average (s) RSD
NOS 125 4653 3.77 988 2.36
cp4-epsps 145 3879 4.23 1125 2.79
35S 158 6732 4.67 1240 1.87
lectin 210 5087 3.87 1312 1.91
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Simulative soybean sample with RRS content of
0.01
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Conclusions

• Labeling based CE-ECL is a sensitive method for
  PCR amplicon detection.
• Amplification efficiency was not decreased after
  ECL labeling.
• The limit of detection is found to be 0.01 with
  35 PCR cycles.

31

• PCR-Free Detection of Genetically Modified
  Organisms Using Magnetic Capture Technology and
  Fluorescence Cross-Correlation Spectroscopy
• Xiaoming Zhou, Da Xing, Yonghong Tan, Wei R. Chen
  
• Plos One. Nov. 2009, Vol.4, Issue 11

32
Objective
To detect and quantify the target DNA using
dual-color fluorescence cross-correlation
spectroscopy
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Fluorescence Correlation Spectroscopy (FCS)

• Powerful method for determining molecular
  interactions in vitro and in vivo
• Time-averaging fluctuation analysis of small
  molecular ensembles
• Fluctuations in the fluorescence signal are
  induced by molecules entering and leaving the
  illuminated region by random diffusive motion

Schwille et.al, BioEssays 24758764
34
What is FCCS?

• Extended version of FCS
• First proposed by Eigen and Rigler in 1994
• Two spectrally different fluorophores
• Two excitation sources
• Two detectors

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Fluorescence correlation Spectrometer
Schwille et al., Biophysics Textbook Online 2001,
133
36
Simultaneous Detection of a Doubly- Labeled
Target at the single-molecule level
Rhodamine Green Detection Channel (527 nm)
Cy5 Detection Channel (666 nm)
Time (s)
Coincident Events
Slide from Dr.Wonbae Lee
37
Cross-correlation Function, Gc (t)
Intensity
time (sec)
Slide from Dr. Wonbae Lee
38
Experimental procedure

• Genomic DNA was extracted using cetyltrimethyl
  ammonium bromide (CTAB) method.
• EcoR V and Hinf I are used to cleave 211 bp
  target from CaMV35S promoter.
• Streptavidin coated magnetic beads were used to
  purify target DNA through biotin-streptavidin
  linkage.
• Purified target DNA was hybridized with two dye
  labeled gene probes.

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PCR-free identification of GMOs by magnetic
capture FCCS
Streptavidin coated magnetic bead
35S promoter DNA
Biotin labeled capture probe
Cy5 labeled gene
RG labeled gene
Magnetic field
40
Results
41
Cross-correlation functions of singly labeled
gene probes
42
Sensitivity and reliability assessment
43
FCCS detection in soybean
GM-soybean with magnetic capture technology
GM-soybean without magnetic capture technology
Non-GM soybean control
44
Comparison between various methods of GMO
detection
Parameter ELISA Southern blot PCR Real time PCR Current assay
Sensitivity Moderate Moderate High High Moderate
Specificity Fair Good Fair Fair Excellent
Assay time 6-8 h 2-4 d 1.5 d 1 d 8-10 h
Potential to be quantitative Yes No No Yes Yes
Needs special equipment Yes Yes Yes Yes Yes
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Conclusions

• A magnetic bead based PCR-free method was
  introduced.
• Sensitivity accuracy of the FCCS system was
  evaluated by PCR.
• Sensitivity of 0.05 nM was observed.

46
Acknowledgements

• Dr. Soper
• Dr. Robin L. Mc Carley
• Soper research group