Lecture 18, Chapter 11

Analysis of transgenic plantspart I

Mat Halter3/27/12

Discussion questions1. What are the established methods to determine if a plant is transgenic and

whether the transgene(s) is expressed?2. In a Southern or northern blot, through what type of chemical bond does

the complementary probe bind to nucleic acid?3. Nucleic acids and proteins are separated according to size in agarose and

sodium dodecyl sulfate–polyacrylamide gel electrophoreisis (SDS-PAGE) gels, respectively. Why do both types of macromolecules migrate toward the anode in an electrical current?

4. What is gene expression, and how can you measure it?5. Explain why phenotypic data provide evidence of transformation but not

proof of a transformation event.6. What factors are most important when designing a Southern blot

experiment to test for transgenic status?

Is my plant transgenic?

P VisualSelection T P Antibiotic

Selection T P Gene of Interest T

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Visual Selection (ex. OFP)

Antibiotic Selection(ex. Hygromycin)

Fluorescent Proteins

http://en.wikipedia.org/wiki/File:FPbeachTsien.jpg

Visual selection using FPsT+ T- T- T+

Antibiotic Selection

• When a mixture of transformed and untransformed callus is placed on antibiotic selection media, only the transformed callus carrying the antibiotic selection cassette is able to survive and grow.

• In most cases, the untransformed callus dies, making it “easy” to select for callus carrying the T-DNA.

Figure 9.3

Sometimes “escapes” occur– for kanamycin resistance markers tissue is red—very stressed

Is my plant transgenic?• Surviving selection—but remember that there can be

escapes. Need more proof besides surviving selection.

• Reporter genes—better. But there must be a reporter gene in the vector.

• All around easy test—PCR. But what if Agrobacterium survives in low amounts in the T0 plants? Could give a false positive band. PCR is ok for biolistics.

• Can do PCR on T1 plants or look at segregation of the transgene.

GFP+Bt segregation

Using GFP screening to “see” Bt when the transgenes are linked. Nat Biotechnol 17:1125

Stable integration of transgene

• Transgene is permanently integrated into the genome of the host plant.

• Transmitted to progeny (Tn plants) in Mendelian fashion

• Need convincing proof of stable integration• Multiple assays are possible—but most

researchers are best convinced by Southern blot data.

Why all the mystique and skepticism?

Good reasons for doubt

• New methods don’t always work, but wishful thinking takes over (see Chapter 10 section—the Rush to Publish)

• Resilient Agrobacterium can linger• The unexpected can be tricky.• Others?

Molecular characterization of transgenic plants

• PCR- Simplest and fastest method. Prone to false positives.

• Southern Blot- Confirms insertion of the tDNA into the genomic DNA of the target organism, as well as provides insertion copy number.

• Northern Blot- Confirms the presence of RNA transcript accumulation from the transgene of interest.

• Western Blot- Confirms presence of the PROTEIN produced from the inserted transgene of interest.

• qRT-PCR- Provides a relative expression level for the gene of interest—transcript—like Northern blot.

PCR and DNA Gel Electrophoresis

PCR- Polymerase chain reaction, uses DNA primers to amplify a target sequence of DNA, producing billions of copies of identical DNA.

Gene cloning Molecular analysis

(Confirmation of the presence of a particular fragment of

DNA in a pool of DNA)

PCR analysis by gel electrophoresis

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Gel electrophoresis

• The migration of DNA through an Agarose matrix using the application of an electric field.

• Agarose, when solidified in a gelatin form, produces a thick netting that allows small particles to move through it quickly, while larger particles move more slowly.

• By moving particles of different size through the agarose gel, they can be separated, with the small particles moving quickly away from the slower moving large particles.

• This method is used to separate DNA fragments by size.

PCR analysis by gel electrophoresis

500 bp

750 bp

1000 bp

1500 bp-

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Ladder Sample

PCR and False Positives

Genomic DNATransgenic plant produced from Agrobacterium-mediated transformation

• In T0 plants, Agrobacterium left over from the initial transformation is still present in all tissues.• Contamination of the genomic DNA with the initial transformation vector that is still present in the agrobacterium can produce a PCR band.

Southern Blot• Southern blotting confirms the presence of

the gene of interest in the genomic DNA of the target plant and avoids the pitfalls of potential false positives.

• Steps– Genomic DNA isolation– Restriction enzyme digestion of genomic DNA– Running digested DNA on agarose gel to separate

fragmented DNA by size. – Transfer of separated DNA to nylon membrane– Hybridization with radioactive DNA probe

Restriction Digestion of Genomic DNA

• Restriction digestion of genomic DNA produces a streak on an agarose gel rather than a single band.

Why?

Example: EcoRI

• What is the probability of a sequence of DNA in a plant genome having the sequence of bases corresponding to an EcoRI cut site?

• Each site can be 4 possible bases (A, T, C, or G), and the EcoRI enzyme requires 6 sites (GAATTC)

• The probability of finding a random site in a genome that happens to have the sequence GAATTC can be calculated:

1⁄4 x 1⁄4 x 1⁄4 x 1⁄4 x 1⁄4 x 1⁄4 = 1⁄4096• Probability states that there will be an EcoRI cut site once

every 4096 bases, purely by chance.

EcoRI example, cont.• The Arabidopsis thaliana genome is roughly 157,000,000 base

pairs in size.157,000,000⁄4096 = 38,330

• Though this value is only based on probability, and therefore may not be the TRUE number of EcoRI cut sites in this genome, it can still accurately be assumed that there are A LOT of cut sites.

• If restriction digested with EcoRI, the arabidopsis genome would be cut into tens of thousands of pieces, all of unique size.

• This is why when you run a sample of digested genomic DNA, you see a streak, rather than a band. The streak shows all sizes of DNA produced by the random assortment of cut sites within the genome.

Digested Genomic• Essentially, every known restriction enzyme will have cut sites in a plant genome. • How can enzyme selection be used to detect copies of an inserted transgene?

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DNA ProbeEcoRI Site

• Single cutting enzymes can be designed into the T-DNA before transformation that will enable proper digestion of the genome as well as a single cut within the T-DNA.

Southern BlottingIsolated genomic DNA from transgenic plant

Restriction enzyme digest

+

-Gel electrophoresis

Southern BlottingTransfer separated DNA from agarose gel to nylon

membrane

Agarose Gel Nylon Membrane

Southern Blotting• The DNA probe is designed to be complimentary to your gene of interest.

•It is synthesized using radioactive phosphorus, which emits a detectable signal. •The complimentary probe will bind (by hydrogen bonding) only to your gene of interest because of the high sequence specificity.

Hybridizing the DNA probe

Southern Blotting

Lane 1- LadderLane 2- Negative ControlLanes 3-8- Experimental Events

• Bands at different places from event to event indicate insertion at different places in the genome.• The number of bands in each well indicates how many insertions there were in each event.

The Final product

Why is a single cut within the T-DNA necessary?

LB RB RBLB RB

EcoRI Site EcoRI Site

If there is no EcoRI site within the tDNA, after digestion with EcoRI these two insertion sites will be indistinguishable from one another after electrophoresis and probing.

Cutting within the T-DNA is necessary to distinguish each and every insertion event. This is VERY important.

Restriction enzyme selection for Southern blotting

Southern blot analysis of transgenic plants with an orange fluorescent protein

Southern analysis of mOrange plants genomic DNA. A. Genomic DNA digested with BamHI, electrophoresed on 1% agarose

Arabidopsis Lane 1. 21-12-3-3 Lane 2. 21-12-6-1 Lane 3. 21-12-6-6 Lane 4. 21-12-7 Lane 5. 21-12-29 Lane 6. 21-12-34 Lane 7. ‘Columbia’ Tobacco Lane 1. 21-12-2-7 Lane 2. 21-12-5-5 Lane 3. 21-12-5-8 Lane 4. 21-12-6-6 Lane 5. 21-12-8-7 Lane 6. ‘Xanthi’ plasmid pMDC32-mOrange (21-12) BamHI digest

B. Transferred to nylon membrane. Hybridization to 32-P labeled probe of mOrange coding sequence. Exposed to phosphor screen 3 weeks.

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Northern Blot• Confirms the presence of mRNA transcripts

transcribed from the gene of interest in the target organism.

• Extremely similar to the Southern blot, but detects RNA instead of DNA.

• Steps:– Isolation of RNA– Running RNA on agarose gel to separate by size– Transfer separated RNA from gel to membrane– Hybridize a radioactive DNA probe to the RNA on the

membrane

Sound familiar?

Northern Blot

RNA loading controls are necessary to ensure an equal amount of RNA is loaded in each well.

No digestion necessary… why is this?

Discussion questions1. What are the established methods to determine if a plant is transgenic and

whether the transgene(s) is expressed?2. In a Southern or northern blot, through what type of chemical bond does

the complementary probe bind to nucleic acid?3. Nucleic acids and proteins are separated according to size in agarose and

sodium dodecyl sulfate–polyacrylamide gel electrophoreisis (SDS-PAGE) gels, respectively. Why do both types of macromolecules migrate toward the anode in an electrical current?

4. What is gene expression, and how can you measure it?5. Explain why phenotypic data provide evidence of transformation but not

proof of a transformation event.6. What factors are most important when designing a Southern blot

experiment to test for transgenic status?