Molecular Analyses

Molecular Analyses 2007-2008 SSR-PCR, Primer Specific PCR & Southern Blot Introduction Transformation and in vitro culture experiments performed in CIBCM laboratories resulted in the regeneration of several plants, presumably transgenic. To confirm their transgenic status and determine if these plants in fact carry the gene used in the biolistics nuclear transformation, vip3A, molecular analyses were performed, such as PCR with specific primers for the identification of the gene and Southern Blot.

Materials and Methods Plant Genomic DNA Isolation The genomic DNA of regenerated plants was isolated using the GenEluteTM Plant Genomic DNA Miniprep Kit, from SIGMA. When using this kit, one should examine the reagents before use, preheat the Water Bath to 65°C, dilute the Wash Solution Concentrate with 95-100% ethanol and preheat the Elution Solution in the Water Bath. The first step of this procedure consisted in the disruption of the cells, grinding plant tissue into a fine powder in liquid nitrogen using a mortar and pestle; 100 mg of the powder were transferred to a microcentrifuge tube. To lyse the cells, 350 µL of Lysis Solution [Part A] and 50 µL of Lysis Solution [Part B] were added to the tube, which was thoroughly mixed by vortexing and inverting. The mixture was incubated at 65°C for 10 minutes with occasional inversion to dissolve the precipitate. After incubation, 130 µL of Precipitation Solution were added to the mixture, to precipitate the debris; this was mixed completely by inversion and placed on ice for 5 minutes. The sample was centrifuged at maximum speed (12,000–16,000 rpm) for 5 minutes to pellet the cellular debris, proteins, and polysaccharides. The debris was filtered by carefully pipetting the supernatant onto a GenElute filtration column (blue insert with a 2 mL collection tube) and centrifuging at maximum speed for 1 minute, thus removing any cellular debris not removed previously. The filtration column was discarded, retaining the

collection tube with the sample, to which 700 µL of Binding Solution were added, mixing thoroughly by inversion. Separately, the Binding Column was prepared by inserting a GenElute Miniprep Binding Column (with a red o-ring) into a provided microcentrifuge tube and adding 500 µL of the Column Preparation Solution. This was centrifuged at 12,000 rpm for 30 seconds to 1 minute and the flow-through liquid was discarded. The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields. After the Binding Column is ready, the previously obtained lysate is loaded onto it, by carefully pipetting 700 µL of the mixture and centrifuging at maximum speed for 1 minute. The flow-through liquid is discarded, retaining the collection tube. Returning the column to the collection tube, the remaining lysate is added onto the column, repeating the centrifugation as above and discarding the flow-through liquid and collection tube. The binding column is placed into a fresh 2 mL collection tube and 500 µL of the diluted Wash Solution are applied to the column. The tube and binding column are centrifuged at maximum speed for 1 minute, the flow-through liquid is discarded and collection tube retained. A second column wash is performed applying another 500 µL of diluted Wash Solution to the column and centrifuging at maximum speed for 3 minutes to dry the column. Finally, the binding column is transferred to a fresh 2 mL collection tube, to which 100 µL of pre-warmed (65 °C) Elution Solution is applied. This is centrifuged at maximum speed for 1 minute. The elution is repeated, obtaining an eluate that contains pure genomic DNA. The plants used to isolate genomic DNA with the previously described procedure are listed in the following Tables (Tables 1 and 2). The samples do not include all the regenerated plants, only those that produced seed and had viable green tissue to perform the DNA isolation.

Table 1. Identification of plant genomic DNA used in PCR with SSR primers and in PCR with specific primers (Experiments 1 and 2) Plant Block

Plant Code

Variety

Gene

II I I I I I I NA NA I I I I I I

2.3.1b 11.1 37.2 32.1 20 26 59 AM1* AM2* 21 11.3 32.3 57 15 49

CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272

Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Witness Witness Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11)

Aclimatation Date 21/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 10/02/2007 10/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007

DNA Isolation Date 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007 26/04/2007

I 37.1 CR-5272 Vip3(11) 14/02/2007 26/04/2007 *Refers to witness (untransformed) plants that did not go through in vitro culture

Eppendorf Tube Code 1 2 3 4 5 6 7 8 A B C D E F G H

Table 2. Identification of plant genomic DNA used in experiment 2 of PCR with specific primers and Southern Blot Plant Block

Plant Code

Variety

Gene

I I I I I I I I I I I I I I I I I

11,1 11,3 17 20a 20b 23,1 23,2 26a 26b 32 32,2 37,1 37,2 38,1 49 57 59

CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272

Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11)

Aclimatation Date 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007 14/02/2007

DNA Isolation Date 30/06/2008 25/06/2008 25/06/2008 25/06/2008 27/06/2008 25/06/2008 25/06/2008 25/06/2008 30/06/2008 25/06/2008 25/06/2008 27/06/2008 27/06/2008 27/06/2008 27/06/2008 27/06/2008 27/06/2008

Eppendorf Tube Code 11,1⋅BI 11,3⋅BI 17⋅BI 20a⋅BI 20b⋅BI 23,1⋅BI 23,2⋅BI 26a⋅BI 26b⋅BI 32⋅BI 32,2⋅BI 37,1⋅BI 37,2⋅BI 38,1⋅BI 49⋅BI 57⋅BI 59⋅BI

Table 2. Identification of plant genomic DNA used in experiment 2 of PCR with specific primers and Southern Blot (Continuation) Plant Block

Plant Code

Variety

Gene

II II II III III III III III III III III III III IV IV IV IV V V V V V V VI VI VI IX IX IX

2,3,1a E2,2 K2,1 10,1 10,2 12 18 30,1 32,4 37,3 37,5 38,2 38,3 E2,1a E2,1d 22,1,1a 22,1,1b 1,2a 1,2d 2,3,1c 2,3,2b 2,3,2g 2,3,2h 10,3 22 30,2 1 9,1 9,2

CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272 CR-5272

Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Witness Witness Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Vip3(11) Witness Witness Witness

Aclimatation Date 21/02/2007 21/02/2007 21/02/2007 29/03/2007 29/03/2007 29/03/2007 29/03/2007 29/03/2007 29/03/2007 29/03/2007 29/03/2007 29/03/2007 29/03/2007 22/03/2007 22/03/2007 22/03/2007 22/03/2007 14/05/2007 14/05/2007 14/05/2007 14/05/2007 14/05/2007 14/05/2007 16/05/2007 16/05/2007 16/05/2007 08/10/2007 08/10/2007 08/10/2007

DNA Isolation Date 27/06/2008 27/06/2008 27/06/2008 27/06/2008 27/06/2008 27/06/2008 27/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 30/06/2008 08/07/2008 08/07/2008 08/07/2008 08/07/2008 08/07/2008 08/07/2008 08/07/2008 08/07/2008

Eppendorf Tube Code 2,3,1a⋅BII E2,2⋅BII K2,1⋅BII 10,1⋅BIII 10,2⋅BIII 12⋅BIII 18⋅BIII 30,1⋅BIII 32,4⋅BIII 37,3⋅BIII 37,5⋅BIII 38,2⋅BIII 38,3⋅BIII E2,1a⋅BIV E2,1d⋅BIV 22,1,1a⋅BIV 22,1,1b⋅BIV 1,2a⋅BV 1,2d⋅BV 2,3,1c⋅BV 2,3,2b⋅BV 2,3,2g⋅BV 2,3,2h⋅BV 10,3⋅BVI 22⋅BVI 30,2⋅BVI 1⋅BIX 9,1⋅BIX 9,2⋅BIX

SSR-PCR (Primer Pair RM-38) The genomic DNA samples described in Table 1 were used for this part of the research, using them in a 1:2 dilution. The pair of SSR primers (RM-38) was used at a concentration of 20 µM. PCR reactions contained the following: 13,45 µl of sterile destilled water, 2,0 µl of PCR Buffer (10X) with KCl, 1,2 µl of MgCl2 (25mM), 0,4 µl of dNTPs (10mM), 0,4 µl of each primer (20 µM), and 0,15 µl of Taq DNA polymerase (5u/µl); 2 µl of genomic DNA (1:2) was added. Each reaction had a total volume of 20 µl. The

temperature profile consisted of 1 min at 95°C, followed by 35 cycles of 1 min at 95°C, 15 s at 55°C, and 30 s at 72°C. The 35 cycles were followed by one cycle of 7 min at 72°C.

Primer Specific PCR Experiment 1: Specific Primers Synthesized on April 2007 (Ordered on November 2006) DNA samples used in this experiment include plant genomic DNA described in Table 1 and plasmid DNA described in Table 3. All samples were used in a 1:2 dilution. Two pair of specific primers were used: one pair was specific to the cry2Ab gene and the other pair was specific to the vip3A gene. All primers were used at a concentration of 20 µM. PCR reactions contained the following: 26 µl of sterile destilled water, 5,0 µl of PCR Buffer (10X) with KCl, 4,0 µl of MgCl2 (25mM), 2,0 µl of dNTPs (10mM), 2,0 µl of each primer (20 µM), and 1,0 µl of Taq DNA polymerase (5u/µl); 2 µl of genomic DNA (1:2) were added. Each reaction had a total volume of 50 µl. The temperature profile consisted of 5 min at 95°C, followed by 30 cycles of 30 s at 95°C, 45 s at 50°C, and 60 s at 72°C. The 30 cycles were followed by one cycle of 10 min at 72°C. Table 3. Plasmid DNA used in PCR reactions and Southern Blot

10 11 7

Eppendorf Tube Code pSCRIPT-Vip3 pSCRIPT-Cry2 pTOPO-Vip3 pTOPO-Cry2 Vip3(10) Vip3(11) Cry2(7)

Dilution used in PCR Reactions 1:2 1:2 1:100 1:100 1:2 1:50 1:2

cry2Ab

12

Cry2(12)

1:100

-

-

pCAMBIA 1305.2

1:100

Plasmid

Gene

Clone

pSCRIPT pSCRIPT pTOPO pTOPO pCAMBIA 1305.2 pCAMBIA 1305.2 pCAMBIA 1305.2

vip3A cry2Ab vip3A cry2Ab vip3A vip3A cry2Ab

pCAMBIA 1305.2 pCAMBIA 1305.2

Experiment 2: Specific Primers Synthesized on January 2008 (Ordered on December 2007) DNA samples used in this experiment include plant genomic DNA described in Table 2 and plasmid DNA described in Table 3. Plant genomic DNA samples were used in a 1:2 dilution, while plasmid DNA samples were used in different dilutions. One pair of specific primers was used (with different sequences from those of experiment 1), specific to the vip3A gene. All primers were used at a concentration of 10 µM. PCR reactions contained the following: 6,5 µl of sterile destilled water, 12,5 µl of PCR Master Mix (2X) [Fermentas], and 1,0 µl of each primer (10 µM); 4 µl of genomic DNA were added. Each reaction had a total volume of 25 µl. The temperature profile consisted of 3 min at 95°C, followed by 30 cycles of 30 s at 95°C, 45 s at 53°C, and 60 s at 72°C. The 30 cycles were followed by one cycle of 10 min at 72°C.

Southern Blot The labeling of the probe used in the Southern Blot and the Southern Blot were performed using the DIG-High Prime DNA Labeling and Detection Starter Kit II, from Roche Molecular Biochemicals. The procedures followed were adapted from the DIG Application Manual for Filter Hybridization from the same manufacturer.

Purification of PCR products for labeling of probe The procedure for labeling of probes required a DNA template; in this case two different PCR products were used as templates: pTOPO-vip3A and pCAMBIA-vip3A. The PCR products had to be purified, in order to be used for the labeling of the probes. The purification was performed following the procedure of the DNA Gel Extraction Kit (Fermentas). The first step to extract the DNA from residual reaction components was an evaluation of the volume of the sample containing DNA, this volume was increased to 100 μL using distilled sterile water. The following step consisted in the addition of 300 μL of Binding solution and 10 μL of the resuspended silica powder suspension. The sample is incubated at 55°C, vortexing every 2 minutes to keep silica powder in suspension.

The silica powder/DNA complex is centrifuged for 5 seconds to form a pellet, and the supernatant is removed. This is continued by three washes of the pellet. Each washing procedure is performed by adding 500 μL of ice cold wash buffer, vortexing and centrifuging for 5 seconds, pouring of the supernatant each time. During each washing the pellet should be resuspended completely. After the supernatant from the last wash was removed, the tube is centrifuged again and the remaining liquid was removed with pipette. The final step of this protocol consisted in the elution of DNA into water or TE buffer. For this, the pellet was resuspended in 100 μL of deionized water or TE and incubated at 55°C for 5 minutes. The tube was centrifuged and the supernatant was transferred into a new tube avoiding the pellet. The elution was performed twice.

Labeling of Probe Two different PCR products were used as templates for the labeling of two probes: pTOPO-vip3A and pCAMBIA-vip3A. Initially, 16 μL of purified PCR product was denatured by heating in a boiling water bath for 10 minutes. This was quickly chilled in an ice bath. After complete denaturation of DNA, 4 μL of DIG-High Prime were added, mixing and centrifuging briefly. The mixture was incubated for 22 hours at 37°C. After the overnight incubation, the labeling reaction was stopped by adding 2 μL of 0.2 M EDTA and heating to 65°C for 10 min.

Estimation of Probe Yield Three working solutions were prepared: 1 ng/μL of each labeled DNA probe and 1 ng/μL of DIG-labeled Control DNA, all diluted with DNA Dilution Buffer. Using the DNA Dilution Buffer, separate serial dilutions of all working solutions were prepared, following Table 4.

Table 4. Serial dilutions of working solutions for the estimation of Probe Yield

A fte

r

preparing the serial dilutions, they were spotted on a narrow strip of Positively Charged Nylon Membrane, by adding 1 μL of probe dilutions to the membrane in two rows. This was performed for both labeled probes and for the control DNA, in separate strips. DNA spots were then fixed to the membrane using a UV light Crosslinker. After the fixation of DNA onto the membranes, a series of washes and incubations with different buffers took place. First, the membranes were rinsed briefly (1-5 min) in Washing Buffer. Then, they were incubated for 30 min in 100 mL of Blocking solution, followed by an incubation for 30 min in 20 mL of Antibody solution. The process continued with two incubations for 15 min each in 100 mL of Washing buffer and an equilibration for 2-5 min in 20 mL of Detection buffer. Finally, membranes were incubated in 10 mL of freshly prepared color substrate solution (10 mL of Detection buffer + 2 μL of Color Substrate Solution) in an appropriate container in the dark, without shaking.

Southern Blot The first step was to separate the DNA samples on an Agarose gel (0,8 %), using PCR products. After the electrophoresis, DNA in the gel was denatured by submerging it in Denaturation Solution (0.5 M NaOH, 1.5 M NaCl) for 15 min at room temperature, with gentle shaking (performed twice). The gel was rinsed with sterile, double distilled water and submerged in Neutralization Solution (0.5 M Tris-HCl, pH 7.5; 1.5 M NaCl) for 15 minutes at room temperature (performed twice). The gel was equilibrated for 10 min in 20X SSC.

The next step consists on setting up the blot transfer, avoiding the formation of air bubbles. First, a piece of Whatman 3MM paper previously soaked with 20X SSC was placed atop a “ bridge” that rests in a shallow reservoir of 20X SSC. The gel was placed atop the soaked sheet of Whatman 3MM paper, rolling a sterile pipette over the sandwich to remove all air bubbles that formed between the gel and paper. A piece of Positively Charged Nylon Membrane was cut to match the size of the gel and it was placed on the DNA-containing surface of the gel, using a pipette to eliminate air bubbles as before. The blot assembly was completed by adding a dry sheet of Whatman 3MM paper, a stack of paper towels, a glass plate, and a 200– 500 g weight. The transfer was completed overnight. The next day, while the blot was still damp, the DNA was fixed to the blot by UV light crosslinking. The membrane was placed (DNA side facing up) on Whatman 3MM paper that had been soaked in 2x SSC, exposed to UV light and allowed to air dry. The membrane was prehybridized with 25 mL of DIG Easy Hyb (Hybridization Buffer), incubating it for 30 min at 35°C. This was followed by a hybridization step, in which the membrane was incubated with the hybridization solution overnight. To prepare this solution, the probe had to be denatured (the probe used was the one prepared from PCR product pTOPO-vip3A). To denature the probe 1,25 μL of it were added to 50 μL of double distilled water, the mixture was placed into a boiling water bath for 5 minutes and chilled quickly in an ice bath. The 51,25 μL of the denatured probe were immediately added to a tube containing 10 mL of prewarmed DIG Easy Hyb to form the hybridization solution. After the overnight hybridization step, several washes and incubations with different buffers were performed. The blot was removed from hybridization solution and placed in a new tube with 100 mL of fresh Low Stringency Buffer (2X SSC containing 0.1% SDS) for 5 min (performed twice). Two more incubations of 15 min each, at 60°C, were performed with 50 mL of preheated High Stringency Buffer (0,5X SSC containing 0.1% SDS). The membrane was rinsed for 2 min in 50 mL of Washing Buffer. Then, it was incubated for 30 min in 50 mL of Blocking solution, followed by an incubation for 30 min in 50 mL of Antibody solution (50 mL of Blocking solution + 5 μL of Anti-DIG alkaline phosphatase). The process continued with two incubations for 15 min each in 50 mL of Washing buffer and an equilibration for 2-5 min in 20 mL of Detection buffer. Finally, the membrane was

incubated in 10 mL of freshly prepared color substrate solution (10 mL of Detection buffer + 2 μL of Color Substrate Solution) in an appropriate container in the dark, without shaking.

Results and Discussion Plant Genomic DNA Isolation All DNA extractions from samples described in Tables 1 and 2 were successful, which can be seen in the several electrophoresis (1%) performed. Figure 1 presents DNA samples described in Table 1, while Figures 2 to 4 present DNA samples described in Table 2. In all figures, bright bands are observed, indicating a high amount of DNA was isolated.

MM 1

2

3

4

5

6

7

8 MM A B

C

D

E

F

G H Va MM

Figure 1. Plant genomic DNA isolation. MM: 1 kb Molecular Marker; 1 through 7 and B to H: possible transgenic plants containing vip3A gene; 8 and A: witness plants; Va: empty lane. This samples coincide with samples described in Table 1.

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 2. Plant genomic DNA isolation. Lanes 1 and 20: MM (1 kb Molecular Marker); Lane 19: V (empty lane); Lanes 2 through 18 and 21 through 23: possible transgenic plants containing vip3A gene. 2: 11,1·BI, 3: 1,3·BI, 4: 17·BI, 5: 20a·BI, 6: 20b·BI, 7: 23,1·BI, 8: 23,2·BI, 9: 26a·BI, 10: 26b·BI, 11: 32·BI, 12: 32,2·BI, 13: 37,1·BI, 14: 37,2·BI, 15: 38,1·BI, 16: 49·BI, 17: 57·BI, 18: 59·BI, 21: 2,3,1a·BII, 22: E2,2·BII, 23: K2,1·BII These samples coincide with samples described in Table 2.

1

2

3

4

5

6

7

8

9

10 11 12

13 14

15 16

17 18

19 20 21 22

1 isolation. 2 3 4 MLanes 5 6 71, 812 and 18: MM (1 kb Molecular Marker); Figure 3. Plant genomic DNA Lane 17: V (empty lane); Lanes 2 through 11, 13, 14 and 19 through 22: possible transgenic plants containing vip3A gene. 2: 10,1·BIII, 3: 10,2·BIII, 4: 12·BIII, 5: 18·BIII, 6: 30,1·BIII, 7: 32,4·BIII, 8: 37,3·BIII, 9: 37,5·BIII, 10: 38,2·BIII, 11: 38,3·BIII, 13: E,2,1a·BIV, 14: E,2,1d·BIV, 19: 1,2a·BV, 20: 1,2d·BV, 21: 2,3,1c·BV, 22: 2,3,2b·BV. Lanes 15 and 16: witness plants. 15: 22,1,1a·BIV, 16: 22,1,1b·BIV. These samples coincide with samples described in Table 2.

Figure 4. Plant genomic DNA isolation. Lane M: 1 kb Molecular Marker; Lanes 1 through 5: possible transgenic plants containing vip3A gene. 1: 2,3,2 g · BV, 2: 2,3,2 h · BV, 3: 10,3 · BVI, 4: 22 · BVI, 5: 30,2 · BVI. Lanes 6 through 9: witness plants. 6: 1 · BIX, 7: 9,1 · BIX, 8: 9,2 · BIX. These samples coincide with samples described in Table 2.

SSR-PCR (Primer Pair RM-38) SSR-PCR with primer pair RM-38 is analyzed in Figure 5. Samples 2, 3 and 6 did not show amplification products, it is possible this was due to reaction conditions, given that in other PCRs there was a product for these samples (pictures not shown). All other samples presented bands of the expected size, some brighter than others. This test was performed to prove the quality of the plant genomic DNA isolated, in order to be sure that it was capable of amplifying in a PCR reaction.

MM 1

2

3 4

5

6

7

8

+

- Va MM MM A B C D E

F G H

+

- Va MM

Figure 5. PCR products generated by primer pair RM-38. MM: 1 kb Molecular Marker (FERMENTAS); 1 through 7 and B through H: possible transgenic plants containing vip3A gene; 8 and A: witness plants; +: positive control (Rice DNA); -: negative control (PCR reaction without DNA); Va: empty lanes. Samples correspond to those described in Table 1.

Primer Specific PCR Experiment 1: Specific Primers Synthesized on April 2007 (Ordered on November 2006) Figures 6 and 7 correspond to PCR reactions performed with specific primer pairs vip3A and cry2Ab, respectively, with samples described in Table 3. In Figure 6 there are amplification products of the expected size in all clones, except for those in lanes 3, 5 and 9. There are amplification products in samples with cry2Ab gene, which should not have MM A 1 band 2 3of 4the 5expected 6 MM size MMin 7lane 8 13 9 can 10 be 11 observed, 12 13 14however, Va MM the sample occurred.

corresponds to plasmid pCAMBIA 1305.2 without the gene of interest, which can indicate some contamination occurred.

Figure 6. PCR products generated by primer pair specific to vip3A. MM: 1 kb Molecular Marker (FERMENTAS); 1: pSCRIPT-Vip3; 2: pTOPO-Vip3; 3: Vip3(10)a; 4: Vip3(10)b; 5: Vip3(11)a; 6: Vip3(11)b; 7: pSCRIPT-Cry2; 8: pTOPO-Cry2; 9: Cry2(7)a; 10: Cry2(7)b; 11: Cry2(12)a; 12: Cry2(12)b; 13: pCAMBIA 1305.2; 14: Negative Control (water); Va: empty lane. Letters a and b indicate repetitions.

In Figure 7 all samples with the cry2Ab gene showed amplification, with bands of the expected size. There was no amplification in lane 13, which was expected. However, samples in lanes 3, 4 and 6 show a band of the expected size, which wasn´t expected, given the samples of these lanes contain the vip3A gene.

1

2

3

4

5

6 MM

7

8

9

10 11

12

13 14

Figure 7. PCR products generated by primer pair specific to cry2Ab. MM: 1 kb Molecular Marker (FERMENTAS); 1: pSCRIPT-Vip3; 2: pTOPO-Vip3; 3: Vip3(10)a; 4: Vip3(10)b; 5: Vip3(11)a; 6: Vip3(11)b; 7: pSCRIPT-Cry2; 8: pTOPO-Cry2; 9: Cry2(7)a; 10: Cry2(7)b; 11: Cry2(12)a; 12: Cry2(12)b; 13: pCAMBIA 1305.2; 14: Negative Control (water); Va: empty lane. Letters a and b indicate repetitions.

Figure 8 corresponds to PCR amplifications with specific primers for vip3A and cry2Ab. The same samples were used for both reactions, which belong to samples described in Tables 1 and 3. There are amplifications of samples containing the vip3A gene with cry2Ab specific primers, which is not expected. All samples, including the negative control, amplified when vip3A specific primers were used, which also was not expected.

Specific Primers for cry2Ab

MM 1 2

3

4

5

6

7

8 V MM

Specific Primers for vip3A

MM 1 2

3

4

5

6 7

8 V MM

Figure 8. PCR products generated by primer pair specific to cry2Ab and vip3A. MM: 1 kb Molecular Marker (FERMENTAS); 1: Vip3(11) Plant a*; 2: Vip3(11) Plant b*; 3: Witness Plant*; 4: pSCRIPTVip3; 5: Vip3(11)b; 6: Cry2(12)b; 7: pCAMBIA 1305.2; 8: Negative Control (water); V: empty lane. * refers to plant genomic DNA samples from Table 1. Samples in lanes 4 through 7 refer to samples from Table 3.

Both pairs of primers amplified samples for both vip3A and cry2Ab genes, which tells us they are not really specific. Given that the negative controls also showed amplification, there can be some doubt as to the handling of the materials, and it becomes possible that some contamination may have occurred. With these results, the decision of designing new primers was taken.

Experiment 2: Specific Primers Synthesized on January 2008 (Ordered on December 2007) PCR products generated by a new pair of vip3A specific primers can be seen in Figure 9. The cry2Ab specific primers were not used because plants obtained were only transformed with vip3A. All samples show an amplification, which was not expected, given that some samples did not contain vip3A (lanes 2, 4 and 5). Negative control does not show any bands, which discards the possibility of contamination of reagents. However, some contamination or mislabeling of samples may have occurred. Samples from lane 1 to 5 were sequenced (data not shown) to determine if vip3A was present. Indeed, all samples contained vip3A gene, which lead us to determine that cry2Ab gene was lost somewhere in the process. 1

2

3

4 5

V

6 V M

Figure 9. PCR products generated by primer pair specific to vip3A. M: 1 kb Molecular Marker (FERMENTAS); 1: pTOPO-Vip3; 2: pTOPO-Cry2; 3: Vip3(11); 4: Cry2(12); 5: pCAMBIA 1305.2; 6: Negative Control (water); V: empty lanes. Samples from Table 3.

An identical PCR reaction with all samples from table 2 was performed, but no amplification occurred (pictures not shown).

Southern Blot Purification of PCR products for labeling of probe Figure 10 presents PCR products generated by vip3A specific primers, which were purified using the DNA Gel Extraction Kit (Fermentas). All purifications resulted in concentrated DNA, except for lane 1. However, given that lanes 1 and 2 are repetitions of the same sample, they were pooled together in one tube. This was also performed for lanes 4 and 5. These purified products were used later on in the labeling of probes

Ma 1

2 4

5 V Mb

Figure 10. PCR products generated by primer pair specific to vip3A, purified. Ma: MassRuler Molecular Marker (FERMENTAS); Mb: 1 kb Molecular Marker (FERMENTAS) 1: pTOPO-Vip3; 2: pTOPO-Vip3; 4: Vip3(11); 5: Vip3(11); V: empty lane. Samples from Table 3.

Labeling of Probe and Estimation of Probe Yield Figure 11 shows a picture of the three working solutions used to determine the yield of labeled probes. In the DIG-labeled control DNA a clear colored spot can be seen up until spot number 6, to the naked eye, spot number 7 was also seen clearly. In the case of pCAMBIA-vip3A labeled probe, spot number 5 was the last one to be seen; in pTOPOvip3A labeled probe spot number 6 was the last one to appear. These results indicate that in pCAMBIA-vip3A labeled probe contained an adequate amount of labeled DNA, which is a

good result, but not the best. The pTOPO-vip3A labeled probe presented the expected amount of labeled DNA, which means it was the best of the two. This last probe was the

CONTROL

one used to perform the hybridization of the Southern Blot.

1

TOPO

CAMBIA

6

2

3

4

7

8

9

5

1

2

3

4

6

7

8

9

1

2

3

4

6

7

8

9

5

Figure 11. Serial dilutions of labeled probes and control DNA spotted on Positively Charged Nylon Membrane. CONTROL: DIG-labeled Control DNA. CAMBIA: probe labeled from pCAMBIA-vip3A sample. TOPO: probe labeled from pTOPO-vip3A sample. The numbers refer to the dilution located at that spot (Table 4).

5

Southern Blot Figure 12 shows the PCR products generated by vip3A specific primers. This agarose gel (0,8%) was used to transfer it´s DNA to a Positively Charged Nylon Membrane for the hybridization with the labeled probe. None of the plant samples (lanes 1 to 6) showed amplification, while the three plasmid samples did show a band of the expected size. In lane 11, pCAMBIA 1305.2 DNA (1:2) was run, new plasmid isolation, it is not the same as the sample used in lane 9. It was not included in the PCR reaction and it was used as a second negative control. M

1

2 3 4

5 6 7 8

9 10 11 V V M

Figure 12. PCR products generated by primer pair specific to vip3A. M: DNA Molecular Weight Marker VI, DIG Labeled. Lanes 1 through 4: possible transgenic plants containing vip3A gene. 1: 26a·BI, 2: 26b·BI, 3: 32·BI, 4: 32,2·BI. Lane 5 and 6: Witness plants. 5: 22,1,1a·BIV, 6: 1·BIX. 7: pTOPO-Vip3; 8: Vip3(11); 9: pCAMBIA 1305.2; 10: Negative Control (water); 11: pCAMBIA 1305.2 DNA 1:2 Dilution*; V: empty lane. Samples of lanes 1 to 6 correspond to those described in Table 2. Samples 7 to 9 correspond to those described in Table 3. * Refers to plasmid DNA, not a PCR product.

After hybridization and chromogenic detection of the probe on the membrane, the obtained result is seen in Figure 13. Both negative controls (lanes 10 and 11) show no band, as well as the plant samples (lanes 1 to 6). The three bands that were observed in the agarose gel can be seen in the hybridization results (lanes 7, 8 and 9). The bands are thick and have shadows going up and down in each lane; this indicates that the concentration is very high and suggests that the amount of PCR product/DNA sample loaded in the agarose gel was too high.

M

1

2 3 4

5 6 7 8

9 10 11 V V M

Figure 13. Southern Blot: Photograph resulting from hybridization of labeled vip3A gene probe. M: DNA Molecular Weight Marker VI, DIG Labeled. Lanes 1 through 4: possible transgenic plants containing vip3A gene. 1: 26a·BI, 2: 26b·BI, 3: 32·BI, 4: 32,2·BI. Lane 5 and 6: Witness plants. 5: 22,1,1a·BIV, 6: 1·BIX. 7: pTOPO-Vip3; 8: Vip3(11); 9: pCAMBIA 1305.2; 10: Negative Control (water); 11: pCAMBIA 1305.2 DNA 1:2 Dilution*; V: empty lane. Samples of lanes 1 to 6 correspond to those described in Table 2. Samples 7 to 9 correspond to those described in Table 3. * Refers to plasmid DNA, not a PCR product.

The results obtained in the Southern Blot confirm that the samples in lanes 7 to 9 contain the vip3A gene. The sample in lane 9, pCAMBIA 1305.2, should not have the gene, which causes some discomfort. Unfortunately we did not possess a positive control for the gene at the time of the experimentation, but it will be performed again with positive controls donated by Dr. Raj Bhatnagar, from the ICGEB, New Delhi, India.