www.eurofins.comwww.biodiagnostics.net

Verification by Alternative Methods:

ELISA, LFS, & DNA

Brenda Johnson

02/11/2016

Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

Ideal Situation

• The event is present, the gene produces the protein in adequate supply to confer herbicide tolerance, and the seedl ing appears as a normal tolerant.

2Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

Corn: Non-trait Examples

3Document name: Verification by Alternative Methods: ELISA, LFS, & DNA File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 01/29/2016

Glufosinate non-tolerant Glyphosate non-tolerant

Non-Trait: Essential structures are present but af fected by specific chemical-physiological interaction

So what if the call isn’t clear?

• Herbicide bioassay measures the response of the see d or plant to an application of herbicide

• Herbicide tolerance is caused by adequate productio n of protein

• Protein is produced only if the gene is present tha t codes for that protein and that gene is expressed

4Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

So what if the call isn’t clear?

5

TRAIT(insect or herbicide resistance)

Plant DNA Plant DNA

Inserted DNA

35s IVS6 Cry 1Ab T-NOS

DNA Testing Methods

Protein Testing Methods

Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

So what if the call isn’t clear?

A Non-Tolerant seedling might:

• Not have the event/gene-no protein is produced that will cause tolerance

• Seedling contains the gene, but it is not turned on so there is no protein produced

• Seedling contains the gene, but the protein is prod uced at such a low level that the seedling appears non-tolerant

• Too much damage to the seedling to identify toleran ce/non-tolerance (Classified abnormal)

6Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

• The event? DNA by PCR

• The gene? DNA by PCR

• The protein? ELISA

• Answer: The reaction of the seed/seedling to the he rbicide

What are we detecting?

7Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

How can we check our calls?

• Lateral Flow Strip

o Wednesday

• ELISA (Enzyme Linked Immunosorbent Assay)

o Wednesday

• DNA Methods

o Monday & Tuesday

8Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

Lateral Flow Strip

• Detect the protein that confers herbicide tolerance or an insect resistance trait present in the same event

9Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

ELISA

• Detect the protein that confers herbicide tolerance or an insect resistance trait present in the same event

10

Positive Controls

Buffer Blanks

Negative Controls

Trait Negative Sample

Trait Negative Sample

Trait Negative Sample

Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

DNA Methods

• Detect an event or gene that codes for the protein that confers herbicide tolerance

• Promoter (CaMV 35s) – Turns gene on

• Regulatory (IVS6 & IVS2) – Regulates gene function

• Gene(s) of Interest (Cry1Ab & pat) – Codes for intend ed trait(s)

• Terminator (T-NOS) – Turns gene off

11

Inserted DNA

Native DNA

P - CaMV 35s IVS6 Cry1Ab T-NOS

Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

patIVS2

A Word of Caution:

• Requires clear understanding of events, genes, mole cular vs. breeding stacks, and the proteins produced

o Use available resources

o http://www.isaaa.org/gmapprovaldatabase/

o http://cera-gmc.org/GmCropDatabaseEvent/

o Many others listed in Seed Technologist Training Manual-Chapter 14

• It is imperative that the correct protein, gene, ev ent, etc. is selected to avoid false verification

12Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016

Verification by Alternative Methods

Questions?

13Document name: Verification by Alternative Methods File name: NA EDR: NA Document owner: Brenda Johnson Last modified on: 02/02/2016