J.R. Simplot Company Characterization of Inserted DNA 1

Appendix 1

Characterization of Inserted DNA

INDEX

Section Page

Index 1

Introduction 2

Results 13

Summary 62

Materials and Methods 63

Conclusions 66

References 68

Certification 69
J.R. Simplot Company Characterization of Inserted DNA 2

INTRODUCTION

Events were evaluated by DNA gel blot analyses to determine the structure and copy number of the 

integrated DNA insert (vector map presented in Figure 1). The descriptions of the genetic elements are 

shown in Table 1. These studies were carried out as parts of the characterization and biosafety 

assessment of the events. 

DNA gel blot analysis was performed using DNA extracted from greenhouse-grown plants and digested

with EcoRV, HindIII, or EcoRI + ScaI. The digested DNA was run with standard DIG-labeled molecular 

weight markers, DIGII and DIGVII, through 0.7% agarose gels in 0.5X TBE buffer and blotted onto Nylon 

Hybond N+ filters. The DNA insert-derived probes used were named ASN (from the Asn1/Ppo5 gene 

silencing cassette), AGP, AGP2 and AGP3 (all from the Agp promoter driving both silencing cassettes), 

GBS (from the Gbss promoter driving both silencing cassettes), and PHL (from the R1/PhL promoter 

silencing cassette) (Figure 2). To facilitate interpretations, the original films were divided into separate 

segments for each digest with the marker lanes copied and boxed.
J.R. Simplot Company Characterization of Inserted DNA 3

Figure 1. Vector pSIM1278

The vector backbone region, on the left, starts at position 9,957-bp and ends at 19,468-bp (= 9,512-bp). The backbone DNA 

consists mainly of bacterial DNA and is intended only to support maintenance of the DNA insert prior to plant transformation. 

The DNA insert region, including flanking Border sequences, is on the right from 19,469-bp to 19,660-bp and from 1-bp to 

9,956-bp, which is a total of 10,148-bp. The DNA insert consists of T-DNA border-like sequences and potato genomic DNA which 

are intended for stable integration into the plant genome.
J.R. Simplot Company Characterization of Inserted DNA 4

Table 1. Genetic elements of the DNA Insert of pSIM1278, from Left Border site to Right Border site

Genetic Element Origin Intended Function Genbank 

Accession 

Number

Start-End 

Point in 

pSIM1278

Reference

1. Left Border (LB) site1Synthetic Site for secondary cleavage to 

release single-stranded DNA insert

from pSIM1278

AY5665552

(bases 1-25)

19,469 –

19,493

van Haaren

et al. 1989

2. DNA flanking the LB sequence S. tuberosum

var. Ranger 

Russet

Supports secondary cleavage at LB AY5665552

(bases 26-

187)

19,494 –

19,655

3. KpnI restriction site S. tuberosum Site for connection of DNA insert

with LB flanking sequence.

AF393847.1 19,656 –1

4. Promoter for the ADP glucose 

pyrophosphorylase gene (pAgp), 1st 

copy

S. tuberosum

var. Ranger 

Russet

One of the two convergent 

promoters that drives expression of 

an inverted repeat containing 

fragments of Asn1 and Ppo5, 

especially in tubers

HM363752 2-2,261

5. Fragment of the asparagine 

synthetase-1 (Asn1) gene (1st copy

antisense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (9) double stranded 

RNA that triggers the degradation of 

Asn1 transcripts to impair 

asparagine formation

HM363759 2,262-2,666 Chawla et 

al. 20123

6. 3’-untranslated sequence of the 

polyphenol oxidase-5 gene (Ppo5) 

(1st copy, in antisense orientation)

S. verrucosum Generates with (8) double stranded 

RNA that triggers the degradation of 

Ppo5 transcripts to block black spot 

bruise development

HM363754 2,667-2,810

7. Spacer-1 S. tuberosum

var. Ranger 

Russet

Sequence between the 1st inverted 

repeats

HM363753 2,817-2,973

8. 3’-untranslated sequence of the 

polyphenol oxidase-5 gene (Ppo5) 

(2nd copy, in sense orientation)

S. verrucosum Generates (6) double stranded RNA 

that triggers the degradation of 

Ppo5 transcripts to block black spot 

bruise development

HM363754 2,974-3,117

9. Fragment of the asparagine 

synthetase-1 (Asn1) gene (2nd copy, 

in sense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (5) double stranded 

RNA that triggers the degradation of 

Asn1 transcripts to impair 

asparagine formation

HM363759 3,118-3,523 Chawla et 

al. 20123

10. Promoter for the granule-bound 

starch synthase (pGbss) gene (1st

copy, convergent orientation relative 

to the 1st copy of pAgp)

S. tuberosum

var. Ranger 

Russet

One of the two convergent 

promoters that drives expression of 

an inverted repeat containing 

fragments of Asn1 and Ppo5, 

especially in tubers

HM363755 3,530-4,215

11. pAgp, 2nd copy S. tuberosum

var. Ranger 

Russet

One of the two convergent 

promoters that drives expression of 

an inverted repeat containing 

fragments of the promoters of PhL 

and R1, especially in tubers

HM363752 4,232-6,491

12. Fragment of promoter for the 

potato phosphorylase-L (pPhL) gene 

(1st copy, in antisense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (16) double 

stranded RNA that triggers the 

degradation of PhL transcripts to 

limit the formation of reducing 

sugars through starch degradation

HM363758 6,492-7,000

13. Fragment of promoter for the 

potato R1 gene (pR1) (1st copy, in 

antisense orientation)

 S. tuberosum 

 var. Ranger 

 Russet

Generates with (15) double 

stranded RNA that triggers the 

degradation of R1 transcripts to 

limit the formation of reducing 

sugars through starch degradation

HM363757 7,001-7,532

14. Spacer-2 S. tuberosum

var. Ranger 

Russet

Sequence between the 2nd inverted 

repeat

HM363756 7,539-7,796
J.R. Simplot Company Characterization of Inserted DNA 5

Table 1 Continued. Genetic elements of the DNA Insert of pSIM1278, from Left Border site to Right Border site

Genetic Element Origin Intended Function Genbank 

Accession 

Number

Start-End 

Point in 

pSIM1278

Reference

15. Fragment of promoter for the 

potato R1 gene (pR1) (2nd copy, in 

sense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (13) double 

stranded RNA that triggers the 

degradation of R1 transcripts to 

limit the formation of reducing 

sugars through starch degradation

HM363757 7,797-8,328

16. Fragment of promoter for the 

potato phosphorylase-L (pPhL) 

gene (2nd copy, in sense 

orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (12) double 

stranded RNA containing this 

sequence will trigger the 

degradation of PhL transcript to 

limit the formation of reducing 

sugars through starch degradation

HM363758 8,329-8,837

17. pGbss (2nd copy, convergent 

orientation relative to the 2nd copy 

of pAgp)

S. tuberosum

var. Ranger 

Russet

One of the two convergent 

promoters that drives expression 

of an inverted repeat containing 

fragments of the promoters of 

PhL and R1, especially in tubers

HM363755 8,838-9,761

18. SacI restriction site S. tuberosum Site for connection of DNA insert 

with RB flanking sequence.

AF143202 9,762 – 9,767

19. DNA flanking the RB sequence S. tuberosum

var. Ranger 

Russet 

Supports primary cleavage at RBLike siteAY5665552

(bases 231-391)

9,771 – 9,931

20. Right Border (RB) sequence1Synthetic Site for primary cleavage to 

release single stranded DNA insert 

from pSIM1278

AY5665552

(bases 392-416)

9,932 - 9,956 van Haaren 

et al. 1989

1

The LB and RB sequences (25-bp each) were synthetically designed to be similar to and function like T-DNA borders from Agrobacterium 

tumefaciens.

2

GenBank Accession AY566555 was revised to clarify the sources of DNA for the Border regions.

3

ASN1 described as genetic elements 5 and 9 is referred to as StAst1 in Chawla et al. 2012.

Figure 2. DNA Insert Probes

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.

Position 1 starts from the first KpnI cutting site in pSIM1278.

Hybridizing bands were based on their approximate sizes and grouped into three different classes (See 

summary explaining band interpretation in Table 2). For clarification purposes, bands in the gels were 

read by aligning the top with the marker band (see line marking top of band in example here). 
J.R. Simplot Company Characterization of Inserted DNA 6

 Original Bands (OBs): Plant sequences that hybridize with a DNA insert probe but are not associated

with the inserted DNA. They are indicative of hybridizing sequences that are present in both 

untransformed and transformed plants (shown as “ ”).

 Internal Bands (IBs): Plant sequences that hybridize with a probe, within the inserted DNA. These 

DNA fragments with predicted sizes are positioned within the DNA insert and indicative of DNA 

integrity (shown as “ ”). Everything is based on 1 copy of the DNA insert so that the expected 

numbers are if we have one copy.

 Junction Bands (JBs): Plant sequences that hybridize with a probe and comprise part of both the

inserted DNA and the insert-flanking DNA. These sequences comprise both part of the DNA insert

and flanking plant DNA, have unique sizes, and are indicative of copy number (shown as “ ”).

Observed and Expected Bands: Differences in the number of observed bands (Obs.) compared to 

expected (Exp.) bands was noted. The data showing more observed bands compared to expected bands

shows us that we have more than one copy or an additional partial copy. If the expected band is missing 

or smaller than the expected size, the integrated DNA insert has a deletion.

In some cases, Southern blots shown in this appendix will use the same probes and enzyme digests as 

found in other studies (for example, see Appendix 3. Evidence for Stability of the Inserted DNA). The 

observations may differ, depending upon the conditions under which the gels were run. Therefore all 

bands described in the tables representing Southern blot data in this appendix refer to the gels 

presented within this appendix.

Summary of Gel Evaluation: In Table 2, we summarized how gels were interpreted to determine 

original bands, along with internal and junction bands of the inserted DNA. In addition, in Figures 3-6

below, we illustrate the restriction sites and where hybridization occurs on the DNA insert for the 

various probes, thus summarizing how bands are identified through a visual illustration. Also included, 

is a summary of the expected number of original bands for all the varieties (Table 3).
J.R. Simplot Company Characterization of Inserted DNA 7

Figure 3. Expected IB and JB of DNA Insert Digested with Various Enzymes and Hybridized with ASN

Probe

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.

Figure 4. Expected IB and JB of DNA Insert Digested with Various Enzymes and Hybridized with AGP 

Probe

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.
J.R. Simplot Company Characterization of Inserted DNA 8

Figure 5. Expected IB and JB of DNA Insert Digested with Various Enzymes and Hybridized with PHL 

Probe

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.
J.R. Simplot Company Characterization of Inserted DNA 9

Figure 6. Expected IB and JB of DNA Insert Digested with Various Enzymes and Hybridized with GBS 

Probe

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.
J.R. Simplot Company Characterization of Inserted DNA 10

Table 2. Band Interpretation in Events

Probe Restriction 

Enzyme

Size (bp) Position on 

DNA Insert

Band 

Type

Expected 

Bands

Notes:

AGP EcoRV Variable Variable OB 3-5 Multiple bands relating to variety

2293 2872-5165 IB 1 Part of PPO/ASN inverted repeat, 

1st GBS promoter and left portion 

of the second AGP promoter

≥1127 LB Flanking 

region to 

AGP 935

JB 1 Indicates at least a portion of the 

left border side of the DNA insert

AGP HindIII Variable Variable OB 3-6 Multiple bands relating to variety

4230 2251-6481 IB 1 PPO/ASN inverted repeat, 1st GBS 

promoter and left portion of the 

second AGP promoter

≥2443 LB flanking 

region to 

2251

JB 1 Indicates at least a portion of the 

left border side of the DNA insert

AGP EcoRI/ScaI Variable Variable OB 3-5 Multiple bands relating to variety

3751 3525-7276 IB 1 Comprising a DNA insert fragment 

consisting of 1st GBS promoter, 

2nd AGP promoter and part of the 

PHL/R1 inverted repeat

≥3717 LB region JB 1 Predicted size indicative of an 

independent insertion comprising 

at least the left border side of the 

DNA insert

ASN EcoRV Variable Variable OB 1-4 Multiple bands relating to variety

712, 2293 2160-2872, 

2872-5165

IB-1,

IB-2

2 Indicative of intact ASN1/ PPO5-

derived inverted repeat and 1st 

GBS promoter and left portion of 

the 2nd AGP promoter

ASN HindIII Variable Variable OB 1-3 Multiple bands relating to variety

4230 2251-6481 IB 1 Indicative of intact ASN1/ PPO5-

derived inverted repeat as well as 

1st GBSs and 2nd AGP promoter

Variable = Size and position of Original bands (OB) are variable depending on potato variety, and position of 

Junction bands (JB) is variable for each event, depending on the DNA insert integration site.
J.R. Simplot Company Characterization of Inserted DNA 11

Table 2 (Continued). Band Interpretation in Events

Probe Restriction 

Enzyme

Size (bp) Position on 

DNA insert

Band 

Type

Expected 

Bands

Notes:

ASN EcoRI/ScaI Variable Variable OB 2-4 Variable

≥3717 Variable JB 1 Indicative of independent insertion 

comprising at least the left border 

side of the DNA insert

GBS EcoRV Variable Variable OB 2-3 Variable

2293 2872-5165 IB 1 Comprising part of PPO/ASN 

inverted repeat, 1st GBS promoter 

and left portion of the second AGP 

promoter

≥3567 Variable JB 1 Indicative of independent insertion 

comprising at least the right border 

side of the DNA insert

GBS HindIII Variable Variable OB 1-2 Variable

4230 2251-6481 IB 1 Comprising intact ASN1/ PPO5-

derived inverted repeat as well as 

1st GBSs and 2nd AGP promoter

≥2163 Variable JB 1 Indicative of independent insertion 

comprising at least the right border 

side of the DNA insert

GBS EcoRI/ScaI Variable Variable OB 2-3 Variable

3751 3525-7276 IB 1 Comprising 1st GBS promoter, 2nd 

AGP promoter and part of the 

PHL/R1 inverted repeat

≥1903 Variable JB 1 Indicative of independent insertion 

comprising at least the right border 

side of the DNA insert

PHL EcoRV Variable Variable OB 3 Variable

≥3567 Variable JB 1 Indicative of independent insertion 

comprising at least the right border 

side of the DNA insert

PHL HindIII Variable Variable OB 3 Variable

1313 6481-7794 IB 1 Comprising part of the PHL/R1 

inverted repeat

≥2163 Variable JB 1 Indicative of independent insertion 

comprising at least the right border 

side of the DNA insert

PHL EcoRI/ScaI Variable Variable OB 1-5 Variable

3751 3525-7276 IB 1 Comprising 1st GBS promoter, 2nd 

AGP promoter and part of the 

PHL/R1 inverted repeat

≥1903 Variable JB 1 Indicative of independent insertion 

comprising at least the right border 

side of the DNA insert

Variable = Size and position of Original bands (OB) are variable depending on potato variety, and position of 

Junction bands (JB) is variable for each event, depending on the DNA insert integration site.
J.R. Simplot Company Characterization of Inserted DNA 12

Table 3. Number of Original Bands (OBs) for Conventional (Wild Type, WT) Varieties

Probe

Restriction 

Enzyme 

Ranger

Russet (F) 

Russet 

Burbank (E) Atlantic (J) G H

AGP EcoRV 3 3 4 4 5

HindIII 4 4 5 3 6

EcoR1/Scal 5 3 4 4 5

ASN EcoRV 4 3 1 2 1

HindIII 3 3 1 1 1

EcoR1/Scal 3 4 2 2 2

PHL EcoRV 3 3 3 3 3

HindIII 3 3 3 3 3

EcoR1/Scal 1 2 5 5 3

GBS EcoRV 3 2 3 2 2

HindIII 2 1 2 1 1

EcoR1/Scal 3 2 3 2 2
J.R. Simplot Company Characterization of Inserted DNA 13

RESULTS

Copy Number and Integrity: Ranger Russet F10

Plants were molecularly analyzed by Southern blot hybridization using DNA extracted from leaves of 

greenhouse-grown plants (see Materials and Methods). F10 contains a single complete copy of the DNA 

insert (Figure 7). This conclusion was deduced from hybridizations shown in Figures 8-11 which are 

summarized in Table 4. At the Left Border site, a single JB was visualized when EcoRV, HindIII and 

EcoRI/ScaI digests were hybridized with the AGP probe. Similarly, hybridization of EcoRI/ScaI digests 

with the ASN probe visualized a single JB. The presence of a single JB at the Right Border side was 

deduced from hybridizations of EcoRV, HindIII, and EcoRI/ScaI digests with the GBS and PHL probes. 

Figure 7. F10 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

Integrity of the DNA insert is indicated by the predicted sizes of IBs visualized in (a) all three digests 

hybridized with the AGP probe, (b) all three digests hybridized with the GBS probe, (c) EcoRV and HindIII 

digests with the ASN probe, and (d) HindIII and EcoRI/ScaI digests hybridized with the PHL probe. 

Furthermore, JBs are larger than the minimum predicted size from internal restriction site to cleavage 

site in the left border (>1.1-kb) as visualized with EcoRV digest hybridized with the AGP probe, and in the 

right border (>1.9-kb) with EcoRI/ScaI digests hybridized with the GBS probe. Moreover, no other band 

indicative of any truncation was observed indicating that the integrated copy was complete. Intactness 

was confirmed by experiments shown in Appendix 4. Junction Analysis and Event-Specific Detection.
J.R. Simplot Company Characterization of Inserted DNA 14

Figure 8. Ranger Russet DNA Hybridization with the AGP Probe

Genomic DNA of Ranger Russet control (RR) and events F10 and F37 were digested and hybridized with

the AGP probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII 

molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all 

three enzymes were on one blot originally, and were separated into three segments (one for each 

digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) are 

indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated 

by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 15

Figure 9. Ranger Russet DNA Hybridization with the ASN Probe

Genomic DNA of Ranger Russet control (RR) and events F10 and F37 were digested and hybridized with

the ASN probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 16

Figure 10. Ranger Russet DNA Hybridization with the PHL Probe

Genomic DNA of Ranger Russet control (RR) and events F10 and F37 were digested and hybridized with

the PHL probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. Partial digestion of EcoRI site due to 

overlapping DNA methylation is marked by an arrow.
J.R. Simplot Company Characterization of Inserted DNA 17

Figure 11. Ranger Russet DNA Hybridization with the GBS Probe

Genomic DNA of Ranger Russet control (RR) and events F10 and F37 were digested and hybridized with

the GBS probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. Partial digestion of EcoRI site due to 

overlapping DNA methylation is marked by an arrow.
J.R. Simplot Company Characterization of Inserted DNA 18

Table 4. Ranger Russet Event F10: DNA Fragments Hybridizing with DNA Insert -Derived Probes

(Summary of observations from Figures 8-11)

Probe Restriction 

Enzyme

Type of Band

OB IB IB JB JB Interpretation

Exp. Obs. Exp. Obs.

AGP

Figure 8

EcoRV 6.7

5.0

1.4

2.3 2.3 1.1+ 2.6 1. Copy number at Left Border side 

Visualization of a single JB in AGP and ASN digests 

indicates that F10 contains a single DNA insert (at the 

Left Border side).

2. Copy number at Right Border side 

Visualization of a single JB in PHL and GBS digests 

indicates that F10 contains a single DNA insert (at the 

Right Border side).

3. Internal DNA insert Integrity 

Integrity of the DNA insert is indicated by the fact that 

the IBs visualized in all DNA digests have nearly the

expected sizes (see note 5 below regarding variations).

See also Figure 40 for 0.7-kb IB.

4. DNA insert Integrity at the extremitiesThe DNA insert

is complete because all JBs are greater than the 

minimum size expected. Closest to JBs are EcoRV 

fragments with AGP probe (LB) and EcoRI/ScaI

fragments with GBS probe (RB).

5. Variation between expected and observed sizes in 

DNA insert. Slight differences were found between 

the observed and expected size of a 3.8-kb internal 

band between restriction sites for EcoR1 at 3,525-bp 

and Sca1 at 7,276-bp. This band was expected with 

the AGP, PHL,and GBS probes with EcoR1 and Sca1, 

but appeared sometimes at 3.6-kb. This may be 

because of differences in gel loading or possibly the 

influence of plant material resulted in faster 

movement within the gel. See further discussion in 

section titled “Additional Studies on DNA insert

Integrity for all Events.”

HindIII 16

8.0

6.5

5.8

4.2 4.2 2.4+ 5.7

EcoRI/ScaI 4.8

2.7

2.2

1.3

1.0

3.8 3.6 3.7+ 3.8

ASN

Figure 9

EcoRV 8.6

4.4

2.5

1.5

2.3,

0.7

2.3,

0.7#

none none

HindIII 3.2

2.9

2.0

4.2 4.2 none none

EcoRI/ScaI 8.2

7.3

4.8

none none 3.7+ 3.9

PHL

Figure 10

EcoRV 22

20

4.7

none none 3.6+ 7.2

HindIII 20

8.6

6.0

1.3 1.3 2.2+ 7.0

EcoRI/ScaI 1.5 3.8 3.6 1.9+ 6.1

GBS

Figure 11

EcoRV 8.6

7.8

7.2

2.3 2.3 3.6+ 6.6

HindIII 1.4

1.3

4.2 4.2 2.2+ 6.6

EcoRI/ScaI 4.0

3.7

3.4

3.8 3.8 1.9+ 6.1

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with the symbol “#” were 

visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe in EcoRV digestion).
J.R. Simplot Company Characterization of Inserted DNA 19

Copy Number and Integrity: Ranger Russet F37

F37 contains a single complete copy of the DNA insert (see Figure 12. F37 Insert). This conclusion was 

deduced from hybridizations shown in Figures 8-11 which are summarized in Table 5. The following 

hybridizations indicated predicted sizes for IBs: EcoRV, HindIII and EcoRI/ScaI digests hybridized with the 

AGP and the GBS probes, EcoRV, HindIII digests hybridized with the ASN probe and HindIII, EcoRI/ScaI I 

digests hybridized with the PHL probe. Integrity of the DNA insert is indicated by the fact that the IBs

visualized in various DNA digests and probes have predicted sizes. 

Figure 12. F37 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

Furthermore, the following hybridizations indicate that JBs are larger than the minimum predicted size 

from internal restriction site to cleavage site in the border: EcoRV, HindIII, and EcoRI/ScaI digests 

hybridized with the AGP probe, EcoRI/ ScaI digests hybridized with the ASN probe; EcoRV, HindIII, and 

EcoRI/ScaI digests hybridized with the PHL and GBS probe. For instance, the JB visualized with the AGP

probe in EcoRV digest is larger than the minimum predicted size from restriction site to cleavage site in 

the left border (>1.1-kb) and GBS probe in EcoRI/ScaI digest is larger than the minimum predicated size 

(>1.9–kb) in the right border. Moreover, no other band indicative of any truncation was observed 

indicating that the integrated copy was complete.

Data were confirmed by sequencing one of the junction fragments shown in Appendix 4. Junction 

Analysis and Event-Specific Detection.
J.R. Simplot Company Characterization of Inserted DNA 20

Table 5. Ranger Russet Event F37: DNA Fragments Hybridizing with DNA Insert -Derived Probes

(Summary of observations from Figures 8-11)

Probe Restriction 

Enzyme

Type of Band

OB IB IB JB JB Interpretation

Exp Obs Exp obs

AGP

Figure 8

EcoRV 6.7

5.0

1.4

2.3 2.3 1.1+ 6.7

(double 

intensity)

1. Copy number at Left Border side 

Visualization of a single JB in AGP and 

ASN digests indicates that F37 contains a 

single DNA insert (at least the Left Border 

side). The double intensity band at 6.7

indicates 1 OB and 1 JB are the same 

size.

2. Copy number at Right Border side 

Visualization of a single JB in most PHL 

and GBS digests indicate that F37 

contains a single DNA insert (at least the 

Right Border side).

3. Internal DNA insert Integrity 

Integrity of the DNA insert is indicated by 

the fact that the IBs visualized in various 

DNA digests have nearly the expected 

sizes (see note 5 below regarding 

variation). See also Figure 40 for 0.7-kb 

IB.

4. DNA insert Integrity at the extremities

The DNA insert is complete because all 

JBs are greater than the minimum size 

expected. Closest to JBs are EcoRV 

fragments with AGP probe (LB) and 

EcoRI/ScaI fragments with GBS probe 

(RB).

5. Variation between expected and 

observed sizes in DNA insert. Slight 

differences were found between the 

observed and expected size of a 3.8-kb 

internal band between restriction sites 

for EcoR1 at 3,525-bp and Sca1 at 7,276-

bp. This band was expected with the 

AGP, PHL,and GBS probes with EcoR1 

and Sca1, but appeared sometimes at 

3.6-kb. This may be because of 

differences in gel loading or possibly the 

influence of plant material resulted in 

faster movement within the gel. See 

further discussion in section titled 

“Additional Studies on DNA insert

Integrity for all Events.”

*Partial digestion of EcoRI site due to overlapping 

methylation marked by arrow (Figures 10, 11).

HindIII 16

8.0

6.5

5.8

4.2 4.2 2.4+ >23

EcoRI/ScaI 4.8

2.7

2.2

1.3

1.0

3.8 3.6 3.7+ 4.0

ASN

Figure 9

EcoRV 8.6

4.4

2.5

1.5

2.3,

0.7

2.3,

0.7#

none none

HindIII 3.2

2.9

2.0

4.2 4.2 none none

EcoRI/ScaI 8.2

7.3

4.8

none none 3.7+ 4.2

PHL

Figure 10

EcoRV 22

20

4.7

none none 3.6+ 8.2

HindIII 20

8.6

6.0

1.3 1.3 2.2+ >23

EcoRI/ScaI 1.5 3.8 3.6 1.9+ 2.4, 2.3*

GBS

Figure 11

EcoRV 8.6

7.8

7.2

2.3 2.3 3.6+ 7.7

HindIII 1.4

1.3

4.2 4.2 2. 2+ >23

EcoRI/ScaI 4.0

3.7

3.4

3.8 3.8 1.9+ 2.4, 2.3*

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with the symbol “#” were 

visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe in EcoRV digestion).
J.R. Simplot Company Characterization of Inserted DNA 21

Copy Number and Integrity: Russet Burbank E12

E12 contains a single complete copy of the DNA insert (see Figure 13). This conclusion was deduced 

from the hybridizations shown in Figures 14-17 which are summarized in Table 6. 

Figure 13. E12 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

DNA insert copy number assessment was based on the fact that single JBs with sizes greater than the 

minimum predicted sizes (from restriction site to border cleavage site) were visualized for both the Right 

Border side and the Left Border side when the various DNA digests were hybridized with AGP, ASN, PHL

and GBS probes. Intactness of the DNA insert was confirmed by visualization of IBs with predicted sizes. 

This is also confirmed by the junction study, which shows both ends of the DNA insert are intact 

(Appendix 4. Junction Analysis and Event-Specific Detection).
J.R. Simplot Company Characterization of Inserted DNA 22

Figure 14. Russet Burbank DNA Hybridization with the AGP Probe

Genomic DNA of Russet Burbank control (EC) and events E12 and E24 were digested and hybridized with

the AGP probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. Partial digestion of EcoRI site due to 

overlapping DNA methylation is marked by an arrow. 

1.95

1.88

1.95

1.88

1.95

1.88
J.R. Simplot Company Characterization of Inserted DNA 23

Figure 15. Russet Burbank DNA Hybridization with the ASN Probe

Genomic DNA of Russet Burbank control (EC) and events E12 and E24 were digested and hybridized with

the ASN probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. 

1.95

1.88

1.95

1.88

1.95

1.88
J.R. Simplot Company Characterization of Inserted DNA 24

Figure 16. Russet Burbank DNA Hybridization with the PHL Probe

Genomic DNA of Russet Burbank control (EC) and events E12 and E24 were digested and hybridized with

the PHL probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. 

1.95

1.88

1.95

1.88

1.95

1.88
J.R. Simplot Company Characterization of Inserted DNA 25

Figure 17. Russet Burbank DNA Hybridization with the GBS Probe

Genomic DNA of Russet Burbank control (EC) and events E12 and E24 were digested and hybridized with

the GBS probe. The enzymes used are indicated below each panel. Size of the DigII and Dig VII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. 

1.95

1.88

1.95

1.88

1.95

1.88
J.R. Simplot Company Characterization of Inserted DNA 26

Table 6. Russet Burbank Event E12: DNA Fragments Hybridizing with DNA Insert -Derived Probes

(Summary of observations from Figures 14-17)

Probe Restriction 

Enzyme

Type of Band (kb)

OB IB IB JB JB Interpretation

Exp. Obs. Exp. Obs.

AGP

Figure 14

EcoRV 5.2

4.4

1.5

2.3 2.3 1.1+ 2.2 1. Copy number at Left Border side 

 Visualization of a single JB with AGP and ASN probes 

indicates that E12 contains a single DNA insert (at least the 

Left Border side).

2. Copy number at Right Border side 

 Visualization of a single JB with PHL and GBS probes 

indicates that E12 contains a single DNA insert (at least the 

Right Border side).

3. Internal DNA insert Integrity 

Integrity of the DNA insert is indicated by the fact that the 

IBs visualized in various DNA digests have nearly the 

expected sizes (see note 5 below regarding variation). See 

also Figure 40 for 0.7-kb IB.

4. DNA insert Integrity at the extremities

No unexpected bands were observed, and all JBs have 

sizes greater than minimum predicted sizes, indicating the 

overall intactness of the DNA insert. Closest to JBs are

EcoRV fragments with AGP probe (LB) and EcoRI/ScaI

fragments with GBS probe (RB).

5. Variation between expected and observed sizes in DNA 

insert Slight differences were found between the observed 

and expected size of a 3.8-kb internal band between 

restriction sites for EcoR1 at 3525-bp and Sca1 at 7276-bp. 

This band was expected with the AGP, PHL,and GBS probes 

with EcoR1 and Sca1, but appeared sometimes at 3.4 to

3.6-kb. This may be because of differences in gel loading or 

possibly the influence of plant material resulted in faster 

movement within the gel. See further discussion in section 

titled “Additional Studies on DNA insert Integrity for all 

Events.”

HindIII 8.4

7.0

6.6

5.8

4.2 4.2 2.4+ 15

EcoRI/ScaI 4.8

4.0

2.3

3.8 3.6 3.7+ 5.3

ASN

Figure 15

EcoRV 8.0

4.4

1.5

2.3

0.7

2.3

0.7#

none none

HindIII 12

3.2

3.0

4.2 4.2 none none

EcoRI/ScaI 9.4

4.9

4.0

2.7

none none 3.7+ 5.8

PHL

Figure 16

EcoRV 20.0

15.0

8.0

none none 3.6+ 4.5

HindIII 18.0

8.8

6.0

1.3 1.3 2.2+ 5.5

EcoRI/ScaI 1.6

1.5

3.8 3.6 1.9+ 3.3

GBS

Figure 17

EcoRV 8.6

7.6

2.3 2.3 3.6+ 4.5

HindIII 1.3 4.2 4.2 2.2+ 5.2

EcoRI/ScaI 4.2

3.2

3.8 3.4 1.9+ 3.0

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with the symbol “#” were 

visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe in EcoRV digestion).
J.R. Simplot Company Characterization of Inserted DNA 27

Copy Number and Integrity: Russet Burbank E24

E24 contains a single, mostly complete, copy of the DNA insert (see Figure 18). This conclusion was 

deduced from the hybridizations shown in Figures 14-17 which are summarized in Table 7.

Figure 18. E24 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

DNA insert copy number assessment was based on the fact that single JBs were visualized for both the 

Right Border side and the Left Border side when the various DNA digests were hybridized with AGP, ASN, 

PHL and GBS probes.

Visualization of IBs with predicted sizes confirmed that the DNA insert was generally intact. However, 

the DNA insert has an approximate 500-bp deletion at its Left Border side because the associated JBs

(3.2–kb) are smaller than the minimum size expected (3.7-kb) when AGP and ASN probes were used for 

hybridization with EcoRI/ScaI digests. Since the AGP probe, which starts from the 5’ end of AGP

promoter, still detected the left side junction signal with lighter intensity, the deletion is within the AGP

probe and does not affect efficacy of the silencing constructs. The Right Border junction was sequenced 

as described in Appendix 4. Junction Analysis and Event-Specific Detection. 
J.R. Simplot Company Characterization of Inserted DNA 28

Table 7. Russet Burbank Event E24: DNA Fragments Hybridizing with DNA Insert -Derived Probes

(Summary of observations from Figures 14-17)

Probe Restriction 

Enzyme

Type of Band (kb)

OB IB IB JB JB Interpretation

Exp. Obs. Exp. Obs.

AGP

Figure 14

EcoRV 5.2

4.4

1.5

2.3 2.3 1.1+ 20.0 1. Copy number at Left Border side 

Visualization of a single JB with AGP and ASN probes 

indicates that E24 contains a single DNA insert (at least Left 

Border side).

2. Copy number at Right Border side 

Visualization of a single JB with PHL and GBS probes

indicates that E24 contains a single DNA insert (at least the 

Right Border side).

3. Internal DNA insert Integrity 

Integrity of the DNA insert is indicated by the fact that the 

IBs visualized in various DNA digests have nearly the 

expected sizes (see note 5 below regarding variation). See 

also Figure 40 for 0.7-kb IB.

4. DNA insert Integrity at the extremities

The DNA insert suffers a small deletion at its Left Border 

extremity because the JBs are smaller than the minimum size 

expected (> 3.7-kb). The deletion is upstream from where 

the AGP probe anneals, which means not greater than a few 

hundred base pairs, and does not affect the functional 

activity of the ASN1/PPO5 gene silencing cassette.

 

 The right side of the DNA insert is intact because all JBs of 

PHL and GBS are greater than the minimum size expected. 

Closest to JBs are EcoRV fragments with AGP probe (LB) and 

EcoRI/ScaI fragments with GBS probe (RB).

5. Variation between expected and observed sizes in DNA 

insert. Slight differences were found between the observed 

and expected size of a 3.8-kb internal band between 

restriction sites for EcoR1 at 3525-bp and Sca1 at 7276-bp. 

This band was expected with the AGP, PHL,and GBS probes 

with EcoR1 and Sca1, but appeared sometimes at 3.4 to 3.6-

kb. This may be because of differences in gel loading or 

possibly the influence of plant material resulted in faster 

movement within the gel. See further discussion in section 

titled “Additional Studies on DNA insert Integrity for all 

Events.”

HindIII 8.4

7.0

6.6

5.8

4.2 4.2 2.4+ 4.6

EcoRI/ScaI 4.8

4.0

2.3

3.8 3.6 3.7+ 3.2

ASN

Figure 15

EcoRV 8.0

4.4

1.5

2.3

0.7

2.3

0.7#

none none

HindIII 12

3.2

3.0

4.2 4.2 none none

EcoRI/ScaI 9.4

4.9

4.0

2.7

none none 3.7+ 3.2

PHL

Figure 16

EcoRV 20.0

15.0

8.0

none none 3.6+ 23.0

HindIII 18.0

8.8

6.0

1.3 1.3 2.2+ 4.7

EcoRI/ScaI 1.6

1.5

3.8 3.6 1.9+ 2.5

GBS

Figure 17

EcoRV 8.6

7.6

2.3 2.3 3.6+ 23.0

HindIII 1.3 4.2 4.2 2.2+ 4.5

EcoRI/ScaI 4.2

3.2

3.8 3.4 1.9+ 2.5

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with the symbol “#” were 

visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe in EcoRV digestion).
J.R. Simplot Company Characterization of Inserted DNA 29

Copy Number and Integrity: Atlantic Event J3

J3 contains one nearly intact and one partial copy of the DNA insert, connected reversely at the left 

border side, with deletions of LB regions and parts of the adjacent AGP promoters. The partial copy 

lacks the 3’ end of the central AGP and the rest of the R1/PHL promoter silencing cassette (see Figure 

19). This conclusion was deduced from Southern hybridizations shown in Figures 20-23 which are 

summarized in Table 8. 

Figure 19. J3 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

DNA insert copy number assessment was based on the fact that 2 JBs were visualized with 

hybridizations for the Left Border side. However, only one IB was observed when the digested DNA was 

hybridized with the AGP probe, indicating that a truncation occurred within the central AGP promoter in 

one copy of the DNA insert. Additional evidence for the occurrence of a truncation comes from the fact 

that only one IB and one JB were visualized from DNA digests hybridized with the PHL probe, which 

means that the truncated DNA insert lacks PHL fragments. Furthermore, only one IB and two JBs rather 

than two IBs and two JBs were visualized with the GBS probe (one IB and one JB per intact DNA insert).

One 1.6-kb EcoRV band and one 4.2-kb HindIII band visualized in the hybridization with AGP probe are 

double intense, suggesting that two copies of the DNA insert can be connected head to head with 

deletion of Left Borders and part of AGP promoters as shown in Figure 19 (J3 Insert). Indeed, 6.6-kb

bands containing AGP or ASN from both DNA insert copies are found in the EcoR1/ScaI DNA digestions 

probed with AGP and ASN, respectively. 

This structure comprising two connected DNA inserts was also confirmed by sequence analysis 

described in Appendix 4. Junction Analysis and Event-Specific Detection.
J.R. Simplot Company Characterization of Inserted DNA 30

Figure 20. Atlantic Hybridization with the AGP Probe

Genomic DNA of Atlantic control (JC) and events J3, J55 and J78 were digested and hybridized with the 

AGP probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 31

Figure 21. Atlantic Hybridization with the ASN Probe

Genomic DNA of Atlantic control (JC) and events J3, J55 and J78 were digested and hybridized with the 

ASN probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 32

Figure 22. Atlantic Hybridization with the PHL Probe

Genomic DNA of Atlantic control (JC) and events J3, J55 and J78 were digested and hybridized with the 

PHL probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 33

Figure 23. Atlantic Hybridization with the GBS Probe

Genomic DNA of Atlantic control (JC) and events J3, J55 and J78 were digested and hybridized with the 

GBS probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). Lanes for all three 

enzymes were on one blot originally, and were separated into three segments (one for each digest) with 

the marker lanes copied and boxed for better illustration. The original bands (OBs) are indicated by “ ”, 

the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated by “ ”. The 

definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 34

Table 8.Atlantic Event J3: DNA Fragments Hybridizing with DNA Insert -Derived Probes

(Summary of observations from Figures 20-23)

Probe Restriction 

Enzyme

Type of Band (kb)

OB IB IB JB JB Interpretation

Exp. Obs. Exp. Obs.

AGP

Figure 20

EcoRV 8.0

5.2

2.1

1.4

2.3 2.3 1.1+ 6.6

1.6*

1. Copy number at Left Border side 

Visualization of two JBs in all DNA digests with the AGP probe 

indicates that J3 contains two DNA insert copies (at least at the Left 

Border side). With the ASN probe, 3 IBs (2.3, 0.7 - double intensity) 

and 1 unexpected JB (6.6) were visualized in the EcoRV digestion. 

With the ASN probe in the HindIII digestion, we visualized 1 IB (4.2) 

and 1 unexpected JB (7.8) containing the ASN inverted repeat, which 

indicates the presence of two DNA insert copies at the LB side. With 

the ASN probe and EcoRV, the presence of a junction band indicates 

there is more than 1 copy. 

2. Copy number at Right Border side

Visualization of a single JB in all DNA digestions with the PHL probe 

indicates that J3 contains one DNA insert at the Right Border side. 

The two JBs in all DNA digests probed with GBS (together with one 

IB) indicates that J3 contains three pGbss elements, 2 in the single 

copy and 1 in the partial copy.

3. Internal DNA insert Integrity 

Only one IB (no double intensity) was found when the three 

digestions (EcoRV, HindIII, EcoRI/ScaI) were probed with AGP and

AGP2 (Figure 38, 2.3-kb), indicating that the second copy of DNA 

insert has a truncation in the internal AGP region. 

The deletion of the internal AGP includes both EcoRV sites (at 5,165 

and 6,390). Additional evidence suggests a deletion of the internal 

AGP based on band intensity which indicates only three rather than 

four 1.2-kb bands in an EcoRV digest hybridized with the AGP3 probe 

(Figure 39). Instead of an expected second 2.3-kb IB in EcoRV digests 

with the ASN probe, we visualized a 6.6-kb fragment, indicating the 

deletion of EcoRV in one internal AGP promoter.

Only one IB was shown in HindIII and EcoRI/ScaI digests hybridized 

with PHL, which indicates the presence of a single copy of DNA 

insert. Thus, the second copy lacks the PHL region. Visualization of 

three bands (1-IB, 2- JBs) hybridizing with the GBS probe indicates J3 

contains three pGbss elements, 2 in the single copy and 1 in the 

partial copy.

4. DNA insert Integrity at the extremities

Both a 1.6-kb EcoRV band and a 4.2-kb HindIII band visualized with 

AGP are double intense, suggesting that two copies of the DNA 

insert are connected reversely at the LB side. In this case, a 2.2-kb 

EcoRV band including LB and partial AGP promoter from both DNA 

insert copies will be expected. However, only a 1.6-kb band was 

observed which means a 0.6- kb deletion from one or both DNA 

inserts occurred during the recombination. The partial DNA insert 

had a deletion at the RB side that extended beyond the first EcoRV 

site (5,165-bp) of the internal AGP promoter.

One JB band in the EcoRI/ScaI digest that is visualized with the ASN 

probe confirmed that the two DNA inserts connected at the LB side. 

When digested with EcoRI/ScaI, we expected double intensity bands 

of AGP and ASN, with the same size of 6.6-kb, and confirmed the 

connection at the LB. 

HindIII 16.0

8.5

7.0

6.0

2.2

4.2 4.2 2.4+ 7.8

4.2*

EcoRI/ScaI 3.9

2.9

2.3

1.1

3.8 3.7 3.7+ 6.6*

2.9*

ASN

Figure 21

EcoRV 1.4 2.3

0.7

2.3

0.7*#

none 6.6

HindIII 3.1 4.2 4.2 none 7.8

EcoRI/ScaI 8.4

7.4

none none 3.7+ 6.6*

PHL

Figure 22

EcoRV 18.0

12.0

7.4

none none 3.6+ 12.0

HindIII 18.0

8.6

6.0

1.3 1.3 2.2+ 12.0

EcoRI/ScaI 1.6

1.5

1.3

1.1

0.9

3.8 3.8 1.9+ 3.8
J.R. Simplot Company Characterization of Inserted DNA 35

Table 8 (Continued). Atlantic Event J3: DNA Fragments Hybridizing with DNA Insert -Derived Probes

(Summary of observations from Figures 20-23)

Probe Restriction 

Enzyme

Type of Band (kb)

OB IB IB JB JB Interpretation

Exp. Obs. Exp. Obs.

GBS

Figure 23

EcoRV 9.2

7.6

7.0

2.3 2.3 3.6+ 12.0

6.1

5. Variation between expected and observed sizes in DNA 

insert. Slight differences were found between the observed 

and expected size of a 3.8-kb internal band between 

restriction sites for EcoR1 at 3,525-bp and Sca1 at 7,276-bp. 

This band was expected with the AGP, PHL,and GBS probes 

with EcoR1 and Sca1, but appeared at 3.7-kb with AGP. This 

may be because of differences in gel loading or possibly the 

influence of plant material resulted in faster movement 

within the gel. See further discussion in section titled 

“Additional Studies on DNA insert Integrity for all Events.”

HindIII 1.3

1.2

4.2 4.2 2.2+ 12.0

8.4

EcoRI/ScaI 4.3

3.8

3.5

3.8 3.8 1.9+ 3.8

2.9

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with “*” were double intensity. 

Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe 

in EcoRV digestion).
J.R. Simplot Company Characterization of Inserted DNA 36

Copy Number and Integrity: Atlantic Event J55

J55 contains one full copy of the DNA insert, and an additional truncated and linked copy that lacks the 

R1/PHL promoter silencing cassette (see Figure 24). This conclusion was deduced from hybridizations 

shown in Figures 20-23 which are summarized in Table 9. 

Figure 24. J55 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

Two observed JBs at the Left Border side in the EcoRV digest hybridized with the AGP probe represented 

two copies of the DNA insert. Indeed, the presence of 2 copies at the Left Border side is confirmed by 

the EcoRI/Scal digest hybridized with the ASN probe. However, only one JB is visualized at the Right 

Border side, indicating that only one copy of the DNA insert has the Right Border region. A truncation 

occurred within the central AGP promoter which removed all sequences at the right of the central AGP

promoter. Also, about 400-bp of the central AGP promoter itself (between the EcoRV and HindIII sites) 

is missing because the 2.3-kb IB visualized when hybridizing an EcoRV digest with the AGP probe is still 

present. Confirmation for the absence of sequences to the right of the central AGP promoter comes 

from the fact that the numbers of IB and JB visualized with the PHL probe are just enough for one intact 

DNA insert copy, indicating that the PHL region in the second copy of DNA insert is missing. Since only 

one JB and two IB identified in an EcoRV digest with GBS probe, the Gbss promoter at the Right Border 

side is not present in one of the DNA inserts.

Furthermore, a 6-kb HindIII JB was shown in all the hybridizations with the AGP, ASN, PHL and GBS

probes, indicating that the RB border of the complete DNA insert is reversely connected to the central 

AGP promoter of the truncated DNA insert. This structure (see Figure 20) is supported by the presence 

of 4.5-kb Scal-EcoRI AGP band and a 4.5-kb Scal-EcoRI GBS band that was double intense because of two 

copies of GBS promoters. This connection of two DNA inserts is also supported by the fact that a 4.5-kb

band connecting 2 DNA inserts was observed when an EcoRV digest was hybridized with AGP3 probe 

(Figure 39). The junction between the two DNA inserts was further confirmed by sequence analyses 

described in Appendix 4. Junction Analysis and Event-Specific Detection.
J.R. Simplot Company Characterization of Inserted DNA 37

Table 9. Atlantic Event J55: DNA Fragments Hybridizing with DNA Insert-Derived Probes

(Summary of observations from Figures 20-23)

Probe Restriction 

Enzyme

Type of Band (kb)

OB IB IB JB JB Interpretation

Exp. Obs. Exp. Obs.

AGP

Figure 20

EcoRV 8.0

5.2

2.1

1.4

2.3 2.3* 1.1+ 5.3

2.0

1. Copy number at Left Border side 

Visualization of two JB in EcoRV digest with AGP probe and EcoRI/ScaI 

digest with ASN probe indicates that J55 contains two DNA inserts (at 

least at the Left Border side).

2. Copy number at Right Border side 

Visualization of a single JB in with the PHL and GBS probes indicates that 

J55 contains one DNA insert at the Right Border side.

3. Internal DNA insert Integrity

There are four bands (IB and JB) found in all three digests probed with 

either AGP or ASN, which means that J55 contains two copies of the 

DNA insert. However, only one IB (4.2) hybridizing with AGP was 

observed in the HindIII digestion, indicating that the associated AGP 

promoter was truncated. The truncated region is about 400-bp with a 

breakpoint between EcoRV (6,390) and HindIII (6,481) sites in the partial 

copy because the 2.3-kb EcoRV IB hybridizing with AGP is still present. 

The number of bands visualized in all three digests probed with PHL 

indicates the presence of one intact copy of the DNA insert (thus, the 

second copy lacks PHL-hybridizing sequences). Visualization of three 

bands in all digests hybridizing with the GBS probe indicates J55 

contains three pGbss elements, 2 in the single copy and 1 in the partial 

copy.

4. DNA insert Integrity at the extremities

In the HindIII digests, a band was shown that ranged from 5.7 to 6.1 in 

the hybridizations probed with AGP, ASN, PHL and GBS. These are all the 

same band but show slight variations with the different probes. This 

suggests the association of two DNA insert copies, whereby the RB 

border of the first copy is linked to a truncated pAGP of the second copy.

The second DNA insert is in the reverse orientation. Evidence for this 

structure comes from the presence of (1) a 4.5-kb Scal-EcoRI band 

hybridizing with AGP, (2) a double-intense 4.5-kb Scal/EcoRI GBShybridizing band, and (3) a double-intense 6.0-kb HindIII GBS band. 

Furthermore, a 4.5-kb band is visualized when EcoRV digest is hybridized 

with the AGP3 probe (Figure 39).

5. Variation between expected and observed sizes in DNA insert. Slight 

differences were found between the observed and expected size of a 

3.8-kb internal band between restriction sites for EcoR1 at 3,525-bp and 

Sca1 at 7,276-bp. This band was expected with the AGP, PHL,and GBS 

probes with EcoR1 and Sca1, but appeared at 3.7-kb with AGP. This may 

be because of differences in gel loading or possibly the influence of 

plant material resulted in faster movement within the gel. See further 

discussion in section titled “Additional Studies on DNA insert Integrity 

for all Events.”

HindIII 16.0

8.5

7.0

6.0

2.2

4.2 4.2 2.4+ 6.7

5.8

3.4

EcoRI/ScaI 3.9

2.9

2.3

1.1

3.8 3.7 3.7+ 5.0

4.5

3.8

ASN

Figure 21

EcoRV 1.4 2.3

0.7

2.3*

0.7#*

none none

HindIII 3.1 4.2 4.2 None 5.8

EcoRI/ScaI 8.4

7.4

None none 3.7+ 5.3

4.0

PHL

Figure 22

EcoRV 18.0

12.0

7.4

None none 3.6+ 4.4

HindIII 18.0

8.6

6.0

1.3 1.3 2.2+ 5.7

EcoRI/ScaI 1.6

1.5

1.3

1.1

0.9

3.8 3.8 1.9+ 4.8

GBS

Figure 23

EcoRV 9.2

7.6

7.0

2.3 2.3* 3.6+ 4.4

HindIII 1.3

1.2

4.2 4.2 2.2+ 6.1*

EcoRI/Sca 4.3

3.8

3.5

3.8 3.8 1.9+ 4.5* 

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with “*” were double intensity. 

Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe 

in EcoRV digestion). 
J.R. Simplot Company Characterization of Inserted DNA 38

Copy Number and Integrity: Atlantic Event J78

J78 contains a single truncated DNA insert (see Figure 25). This conclusion was deduced from 

hybridizations shown in Figures 16-19 which are summarized in Table 10. 

Figure 25. J78 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

Visualization of a single JB when EcoRI, HindII and EcoRI/Scal digests are hybridized with the AGP probe 

demonstrates that J78 contains a single copy of the DNA insert at the Left Border side. However, there is 

a truncation at the Left Border side because the JB is 200-bp shorter than the predicted minimum size 

when EcoRI/Scal digests are hybridized with AGP or ASN probe. Using GBS as a probe, only one IB was 

visualized while the expected JB band on Right Border side was not found in all three digestions, 

indicating that the Gbss promoter adjacent to the Right Border was deleted. PHL hybridization further 

proved that the whole or partial PHL fragment next to Gbss promoter was also missing in both HindIII 

and EcoRI/Scal digestions. Indeed, the junction sequence analysis demonstrated that 486-bp of PHL was 

deleted as described in Appendix 4. Junction Analysis and Event-Specific Detection.
J.R. Simplot Company Characterization of Inserted DNA 39

Table 10. Atlantic Event J78: DNA Fragments Hybridizing with DNA Insert-Derived Probes

(Summary of observations from Figures 20-23)

Probe Restriction 

Enzyme

Type of Band (kb) Interpretation

OB IB 

Exp.

IB 

Obs.

JB 

Exp.

JB

Obs.

AGP

Figure 20

EcoRV 8.0

5.2

2.1

1.4

2.3 2.3 1.1+ 12.0 1. Copy number at Left Border side

Visualization of a single JB in the all digestions with AGP probe 

and EcoR/ScaI with ASN probe indicates that J78 contains one 

copy of DNA insert (at least the Left Border side).

2. Copy number at Right Border side 

J78 contains a single DNA insert without the Right Border 

because only internal PHL and GBS bands were visualized.

3. Internal DNA insert Integrity

Integrity of the DNA insert is indicated by the fact that the IBs

visualized in various DNA digests have nearly the expected 

sizes (see note 5 below regarding variation). See also Figure

40 for 0.7-kb IB.

4. DNA insert Integrity at the extremities

JB bands are about 200-bp shorter than the expected sizes 

when genomic DNA was digested with either HindIII or 

EcoR1/ScaI and probed with either AGP or ASN, indicating that 

there is a truncation at the Left Border region. In addition, J78 

suffers a deletion of about 486-bp comprising PHL and GBShybridizing sequences on the right border side. 

5. Variation between expected and observed sizes in DNA 

insert. Slight differences were found between the observed 

and expected size of a 3.8-kb internal band between 

restriction sites for EcoR1 at 3,525-bp and Sca1 at 7,276-bp. 

This band was expected with the AGP, PHL,and GBS probes 

with EcoR1 and Sca1, but appeared at 3.7-kb with AGP. This 

may be because of differences in gel loading or possibly the 

influence of plant material resulted in faster movement within 

the gel. See further discussion in section titled “Additional 

Studies on DNA insert Integrity for all Events.”

HindIII 16.0

8.5

7.0

6.0

2.2

4.2 4.2 2.4+ 2.1

EcoRI/ScaI 3.9

2.9

2.3

1.1

3.8 3.7 3.7+ 3.5

ASN

Figure 21

EcoRV 1.4 2.3

0.7

2.3

0.7#

None none

HindIII 3.1 4.2 4.2 None none

EcoRI/ScaI 8.4

7.4

None none 3.7+ 3.5

PHL

Figure 22

EcoRV 18.0

12.0

7.4

None none 3.6+ 3.0

HindIII 18.0

8.6

6.0

1.3 1.3 2.2+ none

EcoRI/ScaI 1.6

1.5

1.3

1.1

0.9

3.8 3.8 1.9+ none

GBS

Figure 23

EcoRV 9.2

7.6

7.0

2.3 2.3 3.6+ none

HindIII 1.3

1.2

4.2 4.2 2.2+ none

EcoRI/ScaI 4.3

3.8

3.5

3.8 3.8 1.9+ none

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with the symbol “#” were 

visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe in EcoRV digestion). 
J.R. Simplot Company Characterization of Inserted DNA 40

Copy Number and Integrity: G Event G11

The hybridization data for event G11 are shown in Figures 27-30 and summarized in Table 11. In 

summary, a single JB in AGP and ASN hybridizations indicated that G11 contains a single DNA insert with 

an intact Left Border (See Figure 26). No JB was observed in DNA digests hybridized with the PHL probe 

and EcoRV and HindIII digests hybridized with the GBS probe. This indicated that G11 does not contain 

an insert with a Right Border. Observation of IBs with sizes that are different from the predicted sizes for 

EcoRI/ScaI digests when hybridized with AGP, PHL and GBS probes confirmed that the Right Border side 

of the DNA insert was missing. The truncation occurred on the Right Border side, upstream from the ScaI 

site at position 7,276-bp. A junction study described in Appendix 4. Junction Analysis and Event-Specific 

Detection showed that the actual truncation site was at position 6,618-bp within the DNA insert. A 

summary of the deduced insert for G11 is shown below. Event G11 does not have the pPHL-pR1 inverted 

repeat DNA. The second AGP promoter is immediately upstream from the DNA insert/plant DNA 

junction.

Figure 26. G11 Insert

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII
J.R. Simplot Company Characterization of Inserted DNA 41

Figure 27. G Event DNA Hybridization with the AGP Probe

 EcoRV HindIII EcoRI/SacI

Genomic DNA of G control (GC) and event G11 was digested and hybridized with the AGP probe. The 

restriction enzymes used are indicated below each panel. Size of the DigII and DigVII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb) in gray and black, respectively. 

Lanes for all three enzymes were on one blot originally, and were separated into three segments (one 

for each digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) 

are indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are 

indicated by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. 

1.95

1.88

1.95

1.88

1.95

1.88
J.R. Simplot Company Characterization of Inserted DNA 42

Figure 28. G DNA Hybridization with the ASN Probe

 EcoRV HindIII EcoRI/SacI

Genomic DNA of G control (GC) and event G11 was digested and hybridized with the ASN probe. The 

enzymes used are indicated below each panel. Size of the DigII and DigVII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb) in gray and black, respectively. Lanes for all 

three enzymes were on one blot originally, and were separated into three segments (one for each 

digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) are 

indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated 

by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. 

1.95

1.88

1.95

1.88

1.95

1.88
J.R. Simplot Company Characterization of Inserted DNA 43

Figure 29. G DNA Hybridization with the PHL Probe

 EcoRV HindIII EcoRI/SacI

Genomic DNA of G control (GC) and event G11 was digested and hybridized with the PHL probe. The 

enzymes used are indicated below each panel. Size of the DigII and DigVII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb) in gray and black, respectively. Lanes for all 

three enzymes were on one blot originally, and were separated into three segments (one for each 

digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) are 

indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated 

by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. Observations are 

summarized in Table 8. 
J.R. Simplot Company Characterization of Inserted DNA 44

Figure 30. G DNA hybridization with the GBS probe

 EcoRV HindIII EcoRI/SacI

Genomic DNA of G control (GC) and event G11 was digested and hybridized with the GBS probe. The 

enzymes used are indicated below each panel. Size of the DigII and DigVII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb) in gray and black, respectively. Lanes for all 

three enzymes were on one blot originally, and were separated into three segments (one for each 

digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) are 

indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are indicated 

by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. 

1.95

1.88

1.95

1.88

1.95

1.88
J.R. Simplot Company Characterization of Inserted DNA 45

Table 11. G Event G11: DNA Fragments Hybridizing with DNA Insert-Derived Probes

(Summary of observations from Figures 27-30)

Probe Restriction 

Enzyme

Type of Band (kb)

Interpretation

OB IB IB JB JB

Exp. Obs. Exp. Obs.

AGP

Figure 27

EcoRV 8.4

5.3

2.1

1.5

2.3 2.3 1.1+ 5.3* 1. Copy number at Left Border side

Visualization of a single JB in each of EcoRV digested AGP 

hybridizations and EcoRI/ScaI digested ASN hybridization indicates 

that G11 contains a single DNA insert (containing the Left Border side). 

Although 2 bands were observed in EcoRI/ScaI digested AGP 

hybridization, the absence of an IB in this digest indicates one of the 

bands is the truncated IB. 

*The double intensity band at about 5.3-kb appears to include both

the 5.3-kb OB and the 5.3-kb JB

2. Copy number at Right Border side 

No JB was observed in each DNA digest with PHL hybridization and 

EcoRV, HindIII, or EcoRI/ScaI. Digests with GBS hybridization indicated 

that G11 does not contain the Right Border DNA insert. The missing IB 

from the EcoRI/ScaI digest with GBS hybridization and the visible 5.3-

kb JB is indicative of the truncated IB and confirms there is only one 

copy of the Gbss fragment in the DNA insert at the Left Border side of 

the construct.

3. Internal DNA insert Integrity

IBs with expected sizes observed in EcoRV or HindIII digests with AGP 

and GBS hybridizations indicated the integrity of the DNA insert to the 

second HindIII site (6,481-bp position on pSIM1278). The absence of 

IBs for the EcoRI/ScaI digest with AGP and GBS hybridizations 

indicated that the Right Border side of the DNA insert was truncated at 

a point before the first ScaI site (upstream from 7,276-bp).

4. DNA insert Integrity at the extremities

No IB was visualized in the digest hybridized with the GBS probe and 

EcoRI/ScaI, indicating that the DNA insert for G11 was truncated 

between the HindIII site at 6,481-bp and the Scal site at 7,276-bp. No 

JB was observed when PHL probes for the right border side of the DNA 

insert were used with all three digests. This indicated that a portion of 

the DNA insert was missing from the right border side of the DNA 

insert, including the PHL element. The absence of the IB in the 

EcoRI/ScaI digests probed with PHL suggests that no intact PHL 

sequences are present in the truncated insert.

HindIII 7.0

6.7

6.1

4.2 4.2 2.4+ None

EcoRI/ScaI 4.2

3.0

2.5

1.2

3.8 none 3.7+ 5.9

5.5

ASN

Figure 28

EcoRV 2.5

1.5

2.3

0.7

2.3

0.7#

None None

HindIII 3.1 4.2 4.2 None None

EcoRI/ScaI 7.6

7.0

none none 3.7+ 5.5

PHL

Figure 29

EcoRV 20.0

15.0

8.0

none none 2.2+ none

HindIII 15.0

8.6

5.8

1.3 none 3.6+ none

EcoRI/ScaI 1.7

1.6

1.5

1.3

1.2

3.8 none 1.9+ none

GBS

Figure 30

EcoRV 8.6

7.0

2.3 2.3 3.6+ none

HindIII 1.3 4.2 4.2 2.2+ none

EcoRI/ScaI 4.2

3.4

3.8 none 1.9+ 5.3

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with “*” were double intensity. 

Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe 

in EcoRV digestion). 
J.R. Simplot Company Characterization of Inserted DNA 46

Copy Number and Integrity: H Event H37

The hybridization data for event H37 are shown in Figures 32-35 and summarized in Table 12. Event H37 

contains one complete DNA insert, two truncated inserts, and one additional small fragment only 

comprising a part of the AGP promoter (see Figure 31 below). To simplify explanations, these copies 

have been labeled as copy number 1, 2, 3, and 4 respectively.

Figure 31. H37 Inserts

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

Diagram (4) is a small fragment containing part of the AGP promoter.

At least one copy of the DNA insert is complete as indicated by the IBs and JBs in all the DNA digests that 

have the expected sizes. Two extra JBs (one was expected, 3 total) were observed when EcoRV digests 

were hybridized with the AGP probe, indicating two more DNA insert copies are in H37. One of the 

copies is truncated at the first Asn1 fragment downstream from the Left Border. This is indicated by only 
J.R. Simplot Company Characterization of Inserted DNA 47

one copy of IBs (0.7-kb, 2.3-kb) visualized when the EcoRV digests were hybridized with the ASN probe 

(Figures 33 and 39) and the observation of JBs with EcoRV and HindIII when probed with ASN.

H37 possesses a third partial DNA insert containing the Right Border side. This is indicated by the two 

JBs and two IBs observed in the HindIII digest with the PHL probe. Only one IB (3.7-kb) was observed 

when EcoR1/Scal digests were hybridized with GBS and AGP probes, which indicated that this third DNA 

insert was missing the Left Border region and started at the central GBS near the EcoR1 site, extending 

to the Right Border. 

In addition, a very faint band with a size of about 4.2-kb, indicating the presence a small portion of part 

of an AGP promoter, was detected in H37 with the AGP3 probe (Figure 34). This fragment was not 

visualized with the AGP or AGP2 probe, indicating that the insert itself is smaller than 1.0-kb and has 

been depicted as copy number 4. A similar ~4.2-kb band was identified in event G9, which is not part of 

this submission.

In summary, the deduced inserts for event H37 are shown above. This event is believed to have four 

inserts. One is a full-length DNA insert. The second is a shortened insert with only one AGP promoter 

and a portion of one ASN1 fragment. The third insert is also a shortened DNA insert containing only the 

second inverted repeat with the pPHL and pR1 fragment inverted repeats and the Right Border. The 

fourth insert consists of a part of the AGP promoter only.
J.R. Simplot Company Characterization of Inserted DNA 48

Figure 32. H Hybridization with the AGP Probe

Genomic DNA of H control (Hc) and events H37 and H50 were digested and hybridized with the AGP

probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb) in gray and black, respectively. 

Lanes for all three enzymes were on one blot originally, and were separated into three segments (one 

for each digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) 

are indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are 

indicated by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 49

Figure 33. H Hybridization with the ASN Probe

Genomic DNA of H control (Hc) and events H37 and H50 were digested and hybridized with the ASN

probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb) in gray and black, respectively. 

Lanes for all three enzymes were on one blot originally, and were separated into three segments (one 

for each digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) 

are indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are 

indicated by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 50

Figure 34. H Hybridization with the PHL Probe

 

Genomic DNA of H control (Hc) and events H37 and H50 were digested and hybridized with the PHL

probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb) in gray and black, respectively. 

Lanes for all three enzymes were on one blot originally, and were separated into three segments (one 

for each digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) 

are indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are 

indicated by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 51

Figure 35. H Hybridization with the GBS Probe

 

Genomic DNA of H control (Hc) and events H37 and H50 were digested and hybridized with the GBS

probe. The enzymes used are indicated below each panel. Size of the DIGII and DIGVII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb) in gray and black, respectively. 

Lanes for all three enzymes were on one blot originally, and were separated into three segments (one 

for each digest) with the marker lanes copied and boxed for better illustration. The original bands (OBs) 

are indicated by “ ”, the internal bands (IBs) are indicated by “ ”, and the junction bands (JBs) are 

indicated by “ ”. The definitions for OBs, IBs and JBs can be found in the Introduction. 
J.R. Simplot Company Characterization of Inserted DNA 52

Table 12. H Event H37: DNA Fragments Hybridizing with DNA Insert-Derived Probes

(Summary of observations from Figures 32-35)

Probe Restriction 

Enzyme

Type of Band (kb)

OB IB IB JB JB Interpretation

Exp. Obs. Exp. Obs.

AGP

Figure 32

EcoRV 8.0

7.2

5.4

2.2

1.5

2.3 2.3 1.1+ 3.8

3.2

2.2

1. Copy number at Left Border side

H37 showed three JB bands with the EcoRV and HindIII digests using 

the AGP probe. This indicated three copies of the insert. However, 

the EcoRI/ScaI digest with the AGP probe gave only two JB bands, 

which suggested that one of the three copies was truncated at the 

left border side.

2. Copy number at Right Border side 

H37 contains two copies of the DNA insert extending to the Right 

Border side as viewed with the PHL and GBS enzymes. However, it 

contains three rather than two JBs with EcoRV. The third JB (in bold, 

EcoRV with GBS) is smaller than the minimum size expected, and 

was generated by truncation of one of the two DNA inserts at a site 

within the centrally-located pGbss. 

3. 3. Internal DNA insert Integrity

4. The first DNA insert copy (DNA insert1) is intact because nearly all IBs

visualized in various DNA digests have expected sizes (see comment 

5 below). The second copy (DNA insert2) contains the first AGP 

promoter and is truncated within the Asn1-derived sequence, 

because the expected IBs for a second complete copy were not 

found. Copy number 3, DNA insert3, is truncated as it misses the 

Left Border side, and lacks additional IBs (2.3, 0.7) associated with 

the ASN fragments when digested with EcoRV and hybridized with 

the ASN probe.

5.

6. 4. DNA insert Integrity at the extremities

DNA insert1 is complete because the JBs are greater than the 

minimum size expected. Evidence for the structure of DNA insert2 

were the JBs found with EcoRV and HindIII digests with the ASN 

probe when none are expected. DNA insert2 is truncated at Asn1 as 

indicated by visualization of a JB band that was unexpected and 

smaller in size (2.4) with the ASN probe in the EcoRI/ScaI digest. 

The total size of this copy should be smaller than 2.4-kb since 

EcoRI/ScaI would cut outside of the proposed DNA insert2. DNA 

insert 3 is missing the left side as indicated by a smaller than 

expected band (3.2-kb, in bold) in the EcoRV digestion with GBS 

probe. Also, DNA insert 3 has one IB band plus 3 JB bands in other

digestions with EcoRI/ScaI, and both GBS and PHL probes. These 3-

JBs that hybridize with the PHL and GBS probes are found instead of 

the expected IBs, indicating a truncation to the right of the EcoR1 

site at 3,525-bp.

5. Variation between expected and observed sizes in DNA insert. 

Slight differences were found between the observed and expected 

size of a 3.8-kb internal band between restriction sites for EcoR1 at 

3,525-bp and Sca1 at 7,276-bp. This band was expected with the 

AGP, PHL,and GBS probes with EcoR1 and Sca1, but appeared 

sometimes at 3.7-kb. This may be because of differences in gel 

loading or possibly the influence of plant material resulted in faster 

movement within the gel. See further discussion in section titled 

“Additional Studies on DNA insert Integrity for all Events.”

HindIII 12.0

8.6

7.0

6.6

6.1

5.8

4.2 4.2 2.4+ 20

5.8

3.3

EcoRI/ScaI 4.9

4.3

3.0

1.3

1.1

3.8 3.7 3.7+ 5.8

3.5*

ASN

Figure 33

EcoRV 1.4 2.3

0.7

2.3

0.7#

none 3.3

HindIII 3.4 4.2 4.2 none 6.4

EcoRI/ScaI 9.4

8.6

none none 3.7+ 3.7

2.4 

(weak)

PHL

Figure 34

EcoRV 20.0

14.0

8.2

none none 3.6+ 4.7*

HindIII 16.0

9.2

6.5

1.3 1.3* 2.2+ 6.5

3.3

EcoRI/ScaI 2.4

1.8

1.7

3.8 3.7 1.9+ 5.8

2.4

2.1

GBS

Figure 35

EcoRV 12

8.4

2.3 2.3 3.6+ 4.7*

3.2

HindIII 0.9 4.2 4.2 2.2+ 6.5

6.1

3.3

EcoRI/ScaI 4.3

3.6

3.8 3.8 1.9+ 3.5

2.4

2.1 

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with “*” were double intensity. 

Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe 

in EcoRV digestion). 
J.R. Simplot Company Characterization of Inserted DNA 53

Copy Number and Integrity: H Event H50

The hybridization data for event H50 are shown in Figures 32-35 and summarized in Table 13. Event H50

contains one complete DNA insert as well as one truncated copy (see Figure 36 below). 

Figure 36. H50 Inserts

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

The presence of two JBs at the left border side was observed in all the digests hybridized with the AGP

probe and in the EcoRI/ScaI digest hybridized with the ASN probe. These results suggested two inserts 

with a Left Border side. However, all the digests probed with GBS indicated the presence of a single copy 

containing a Right Border side. One of the inserts is complete, because all the expected IBs were 

present and because the JBs identified were greater than the minimum size expected. However, no IBs

were observed in the other DNA insert copy when HindIII and EcoR1/Scal digests were hybridized with 

PHL probe, indicating that part of pPHL/pR1 inverted repeat was deleted. This analysis was confirmed by 

sequencing the junction fragments (see Appendix 4. Junction Analysis and Event-Specific Detection). 
J.R. Simplot Company Characterization of Inserted DNA 54

Table 13. H Event H50: DNA fragments Hybridizing with DNA Insert-Derived Probes

(Summary of observations from Figures 32-35)

Probe Restriction 

Enzyme

Type of Band (kb)

OB IB IB JB JB Interpretation

Exp. Obs. Exp. Obs.

AGP

Figure 32

EcoRV 8.0

7.2

5.4

2.2

1.5

2.3 2.3* 1.1+ 4.0

1.9

1. Copy number at Left Border side

Visualization of two JBs in three DNA digests with the AGP probe and 

EcoRI/ScaI with the ASN probe indicated that H50 contained two 

copies of the DNA insert comprising the Left Border side.

2. Copy number at Right Border side

Visualization of a single JB in most digests with the PHL and GBS 

probes indicated that H50 contained at least one DNA insert

comprising the Right Border side. The two JB bands observed with

the HindIII digestion and PHL probe indicated a second insert copy 

with a truncation on the RB side.

3. Internal DNA insert Integrity

*The double intensity IB bands observed with the AGP, ASN and 

GBS probes indicated the presence of two copies of the DNA insert. 

However, only single intensity IB bands were observed with the PHL 

probe. This suggests that at least one copy of the DNA insert is 

complete as indicated by the IBs having the expected sizes. The 

second DNA insert copy has a complete cassette on the LB side (see 

double intensity IBs with AGP, ASN and GBS probes and EcoRVand 

HindIII) and truncation on the RB side (seen with the PHL probe).

4. DNA insert Integrity at the extremities

A total of four IB and JB bands with the AGP and ASN probes and 

three IB and JB bands with the PHL and GBS probes were found in 

the H50 event digests. One of the inserts is complete because the 

JBs are greater than the minimum size expected. The second insert 

was truncated (between the HindIII site at 6,481 and the Sca1 site at 

7,276) and the deleted region included the pPHL and pR1 inverted 

repeat, Gbss promoter and Right Border.

5. Variation between expected and observed sizes in DNA insert. 

Slight differences were found between the observed and expected 

size of a 3.8-kb internal band between restriction sites for EcoR1 at 

3,525-bp and Sca1 at 7,276-bp. This band was expected with the 

AGP, PHL,and GBS probes with EcoR1 and Sca1, but appeared 

sometimes at 3.7-kb. This may be because of differences in gel 

loading or possibly the influence of plant material resulted in faster 

movement within the gel. See further discussion in section titled 

“Additional Studies on DNA insert Integrity for all Events.” In the 

EcoR1/Sca1 digest we expected two JBs that were the same size, 

however there was slight variation with one of the band sizes 

specifically between the AGP and ASN probes. 

HindIII 12.0

8.6

7.0

6.6

6.1

5.8

4.2 4.2* 2.4+ 4.7

3.6

EcoRI/ScaI 4.9

4.33.0

1.3

1.1

3.8 3.7 3.7+ 5.7

4.3*

ASN

Figure 33

EcoRV 1.4 2.3,

0.7

2.3*

0.7#*

none none

HindIII 3.4 4.2 4.2* none none

EcoRI/ScaI 9.4

8.6

none none 3.7+ 6.1

4.2

PHL

Figure 34

EcoRV 20.0

14.0

8.2

none none 3.6+ 6.9

HindIII 16.0

9.2

6.5

1.3 1.3 2.2+ 5.7

4.3

EcoRI/ScaI 2.4

1.8

1.7

3.8 3.7 1.9+ 3.7

GBS

Figure 35

EcoRV 12.0

8.4

2.3 2.3* 3.6+ 6.9

HindIII 0.9 4.2 4.2* 2.2+ 5.7

EcoRI/ScaI 4.3

3.6

3.8 3.8 1.9+ 4.3

3.7

The definition of OB, IB, and JB can be found in the Introduction. Bands indicated with “*” were double intensity. 

Bands indicated with the symbol “#” were visualized in a blot shown in Figure 40 (for 0.7-kb IB with the ASN probe 

in EcoRV digestion). 

Additional Studies on DNA Insert Integrity for All Events
J.R. Simplot Company Characterization of Inserted DNA 55

In some cases, minor differences were observed between expected sizes of internal bands and what we 

observed. In multiple events, an internal band was observed of about 3.6-kb in size where we knew the 

expected size to be 3,751 or about 3.8-kb. This band occurs between restriction sites for EcoR1 at 

3,525-bp and Sca1 at 7,276-bp and was expected to be visualized with the AGP, PHL, and GBS probes 

with EcoR1 and Sca1 digests. The difference of about 0.15-kb between observed and expected sizes

most likely was related to small differences in gel loading or the influence of plant tissues that would not 

be part of the marker lanes. The largest difference between expected and observed values was found 

for events E12 and E24. The same sequence of DNA insert containing the second AGP promoter

(expected size was 3.8-kb with EcoR1 and ScaI) was studied using the HindIII digests. For example, 

expected bands with the AGP, ASN, and GBS probes of 4.2-kb between 2,251 and 6,481-bp all include 

the AGP promoter region. For all three probes, the observed bands matched the size of the expected 

bands for both events E12 and E24 (Tables 6 and 7). In addition, the 1.3-kb band between 6,481 and 

7,794-bp restriction sites for HindIII, was observed with the PHL probe in both E12 and E24. Thus, 

additional probes and restriction digests support the observation that the DNA insert in the region of 

the second APG promoter was intact.

To investigate the possibility of a truncation, we conducted studies with probes that would anneal to 

the AGP promoter in that region. Further evidence for the structure of the various inserts was obtained 

by performing additional studies using a new probe that was designed to anneal to the downstream part 

of the AGP promoter. This 402-bp probe, AGP2, was amplified with primers HY1659 and HY1660 (Figure 

37 and Table 14 in Materials and Methods). As shown in Figure 38, hybridization with AGP2 visualized 

an expected 2.3-kb band in DNA of all events, indicating that no AGP promoter residing within the DNA 

insert was truncated in this region. A 999-bp probe, AGP3 (Figure 39), was amplified with primer HY1661 

and HY2662 (Figure 37 and Table 14 in Materials and Methods) and specifically targeted the AGP

promoter region between the two EcoRV digests with an expected size of 1,225-bp. As shown in Figure 

39, the J55 and H37 events each had extra hybridizing DNA fragments in addition to the 1,225-bp 

fragment. This indicated that an AGP promoter residing within one of the DNA inserts of these events 

was truncated at this range.

A short electrophoresis run of EcoRV digests probed with ASN confirmed the presence of the relatively 

small internal EcoRV fragments with an expected size of 0.7-kb (Figure 40). The presence of this band 

provides additional proof of the integrity of the DNA inserts.

Even more evidence exists for the completeness of the DNA insert in the region of the AGP promoter 

(3,525 to 7,276-bp) in the gels used for stability testing (See Appendix 3, Evidence for stability of the 

inserted DNA). For events F10 and F37 (Evidence for stability of the inserted DNA : Figures 4, 5, and 6), 

the gel shows the 2.3-kb section of DNA insert that hybridizes with the AGP probe between the EcoRV

restriction sites at 2,872 and 5,165-bp. This same 2.3-kb band is visualized for events E12 and E24

(Evidence for stability of the inserted DNA : Figures 11, 12, and 13), J3, J5, and J78 (Figures 18, 19, and 

20), G11 (Figures 25, 26, and 27) and H37 and H50 (Figures 33, 34, and 35). Thus, the stability studies 

provide additional evidence that the DNA insert is intact in this region.
J.R. Simplot Company Characterization of Inserted DNA 56

Figure 37. Expected IB and JB of DNA Insert Digested with Various Enzymes and Hybridized with (A) 

AGP2 and (B) AGP3 probes.

(A)

(B)

RV=EcoRV

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.
J.R. Simplot Company Characterization of Inserted DNA 57

Figure 38. Hybridization of EcoRV Digests with AGP2 Probe

Genomic DNA was digested with EcoRV and hybridized with the AGP2 probe. Samples were loaded 

following the variety order E (Russet Burbank), F (Ranger Russet), J (Atlantic), G (G) and H (H). EC, FC, JC, 

GC and HC are wild type control samples for each variety. Size of the DigII and Dig VII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb). Events G9 and E67 are not part 

of this submission.
J.R. Simplot Company Characterization of Inserted DNA 58

Figure 39. Hybridization of EcoRV Digests with AGP3 Probe

Genomic DNA was digested with EcoRV and hybridized with the AGP3 probe. Samples were loaded 

following the variety order E (Russet Burbank), F (Ranger Russet), J (Atlantic), G (G) and H (H). EC, FC, JC, 

GC and HC are conventional variety control samples for each variety. Size of the DigII and Dig VII 

molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). A boxed 

segment of the blot is shown again below the blot with different brightness and contrast to highlight the 

4.2-kb bands in J55, G9, and H37. Events G9 and E67 are not part of this submission.
J.R. Simplot Company Characterization of Inserted DNA 59

Figure 40. Visualization of an Expected 0.7-kb Band in All Events when Hybridized with the ASN Probe

(in addition to previously observed bands in shorter runs)

Genomic DNA was digested with EcoRV and hybridized with the ASN probe. Samples were loaded 

following the variety order E (Russet Burbank), F (Ranger Russet), J (Atlantic), G (G) and H (H). EC, FC, JC, 

GC and HC are wild type control samples for each variety. Size of the DigII and Dig VII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb). Events G9 and E67 are not part 

of this submission.
J.R. Simplot Company Characterization of Inserted DNA 60

PCR Confirmation of EcoR1/Scal Fragments in E, F J, and H lines

Minor differences were observed in some cases between expected sizes of internal bands and what we 

actually observed. The description and figures below clarify why we observed the minor differences.

When the plant genomic DNA is digested with EcoR1 and Scal a 3751-bp internal fragment of the DNA 

insert is expected in all events except G11. In addition to Southern blot analysis, a PCR approach was 

used to further ensure the completeness of the fragment. As shown in Figure 41, four primers were 

designed to amplify two fragments: (A) a 1653-bp fragment including EcoR1 site (3525) and EcoRV site 

(5165); (B) a 2125-bp fragment from EcoRV site (5165) to Scal site (7276). Since the component targeted 

by a forward primer was different from one targeted by a reversed primer, no fragments were expected 

to be amplified from untransformed parent controls. The pSIM1278 plasmid DNA was also used as a 

positive control to amplify the fragment A and B. The results were shown in Figure 42. Both fragment A 

and fragment B were amplified from transformed events but not from untransformed controls. The sizes 

of the two fragments are the same as the expected and the positive controls. We can conclude that the 

3751-bp internal fragment of DNA insert exists in events E12, E24, F10, F37, J3, J55, J78, H37, and H50.

Figure 41. The primers and PCR fragments to confirm 3751-bp EcoR1/Scal fragment of DNA insert

A

B

Fragment Forward primer Reverse primer Length

A CGAATTCGTGATGTGTGGTC GGATGTATGATATCACATGTGTTTG 1653 bp

B CAAACACATGTGATATCATACATCC ACTATTGCAAGAAAATATCAAAGGC 2125 bp

3523-5175

5151-7275

3751 bp
J.R. Simplot Company Characterization of Inserted DNA 61

Figure 42. Confirmation of a 3751-bp internal fragment of DNA insert in E, F, J, and H lines

Bands on the ethidium bromide-stained gels are indicative for the presence of fragment A and B. Data 

for all events, except for G11, are shown. M = one kb DNA marker lane (invitrogen), PC = positive 

control, wt = untransformed recipient variety (“wild type”, the negative control).
J.R. Simplot Company Characterization of Inserted DNA 62

SUMMARY

Events F10, F37, E12, and E24 contain a single DNA insert comprising all sequences needed for 

expression of both the Asn1/Ppo5 gene silencing cassette and R1/PhL promoter silencing cassette.

Events J78 and G11 contain a single DNA insert comprising the sequences needed for expression of the 

Asn1/Ppo5 gene silencing cassette. These two events do not contain all sequences required for 

expression of the R1/PhL promoter silencing cassette.

Events J3, J55, and H50 contain 2 copies of the DNA insert, one of which is full and the other one lacking 

at least part of the R1/PhL promoter silencing cassette.

Event H37 contains one full copy of the DNA insert and three additional DNA insert fragments.
J.R. Simplot Company Characterization of Inserted DNA 63

MATERIALS AND METHODS

Plant Material. Plants used for DNA gel blot analysis were grown for two months in Sunshine mix-1 

(www.sungro.com) in two-gallon pots in a greenhouse that was controlled for temperature (18°C 

minimum/27°C maximum) and light (16-h photoperiod with an intensity of ~1500 μmol/m2/s).

DNA Isolation. For plant DNA isolation, 1.0 g of young potato leaves was ground into a fine powder in a 

mortar using liquid nitrogen. The ground tissue was transferred to a pre-cooled 15 ml conical tube with 

a pre-cooled spatula and stored at -80oC until ready to process. 10 ml extraction buffer (0.35 M 

Sorbitol, 0.1 M Tris-HCl, pH8.0, 0.05M EDTA) was added to, and mixed with, the powder, and the 

resulting suspension was centrifuged at 3,000 rpm for 15 min at room temperature. The pellet was 

thoroughly re-suspended in 2 ml extraction buffer containing 200 μg RNase A. After incubating the 

resuspended DNA at 650C for 20 min with 2 ml of nuclear lysis buffer (0.2 M Tris-HCl pH7.5, 0.005 M 

EDTA pH 8.0 and 20 mg/ml CTAB Hexadecyl Trimetyl Ammonium Bromide) and 800 μl of 5% Sarcosyl, it 

was mixed with an equal volume of chloroform : isoamyl alcohol (24:1), vortexed for about 1 min, and 

centrifuged at 3000 rpm for 5 min at room temperature. The DNA was precipitated with an equal 

volume of isopropyl alcohol, washed with 70% ethanol, air dried, and dissolved in 400-700 μl 1X 

Tris/EDTA buffer (TE). DNA concentration was measured using a spectrophotometer at an OD of 260 nm, 

whereas quality was confirmed by running the DNA on a 0.8% agarose gel in 1X Tris/Acetate/EDTA (TAE)

for 30-40 min at 80 volts. In some cases, an alternative method was used to isolate DNA. The ground 

tissue from 0.7 g young leaves was mixed with 7 ml CTAB buffer (2% CTAB, 1.4 M NaCl, 0.1 M pH 8.0 

Tris-HCI, 20 mM pH8.0 EDTA, 100 μg/ml RNase) and incubated at 55o-65oC for 30 min followed by 

centrifugation at 3,000 rpm for 15 min. DNA was extracted twice with equal volumes of chloroform: 

isoamyl alcohol (24:1) by shaking for 10 min and centrifuged at 3000 rpm for 5 min at room 

temperature. The DNA was precipitated with equal volumes of ethanol and rinsed with 70% ethanol. 

The resulting pellet was dried in air and dissolved in TE buffer. The quality and concentration of DNA was 

determined spectrophotometrically and by running a sample on a 0.8% agarose gel. Control and event 

DNA was always extracted with the same method for a specific comparison.

DNA Gel (Southern) Blot Analysis. 10 μg of plant DNA was digested overnight in 400 μl final volume 

with at least 5 μl (10 units/μl) restriction enzyme (Invitrogen) at 37oC. Digested DNA was concentrated 

by ethanol precipitation (40 μl of 3M NaOAc, pH5.3 and 1 ml ethanol) at -80oC for 10 min followed by a 

wash with 70% ethanol. The DNA pellet was dissolved in 20 μl 1X TE followed by addition of 2 μl DNA 

gel loading buffer, which consists of 40% sucrose and 0.35% Orange G (Sigma) in water.

Membrane Preparation. Digested plant DNA was loaded on a large 0.7% agarose gel (170 ml) containing 

0.5X tris-borate-EDTA (TBE) buffer and 3-5 μl ethidium bromide (10 mg/ml) and run at 25 volts for 18 

hrs. The gel was photographed using a gel documentation system from Alpha Innotech (Santa Clara, 

California), and then depurinated by submerging it into 0.25 N HCl for 20 min. After subsequent 

denaturing in 0.5 M NaOH /1.5 M NaCl for 2X15 min and neutralization in 1.5 M NaCl and 0.5 M Tris-HCl, 

pH7.5, for 2 X 15 min on a shaker at room temperature and equilibrated with 10X SSC for 10 min. The 

transfer of DNA to the nylon membrane was carried out using 10X SSC according to a standard capillary 

transfer method. 

Probe Preparation. The labeling of the PCR-derived probe was achieved using Hotmaster taq enzyme 

and buffer (Fisher BioReagents) according to Roche’s DIG labeling instructions. A standard 50 μl 

reaction consisted of 5 μl 10 x Hotmaster Taq Buffer, 2-5 μl 10 uM forward primer, 2-5 μl 10 μM reverse 

primer, 5 μl DIG labeled dNTP (Roche), 10 ng plasmid template, 0.75 μl Hotmaster Taq polymerase, and 
J.R. Simplot Company Characterization of Inserted DNA 64

water. The PCR amplification conditions were dependent on each DIG-labeled probe. PCR with regular 

dNTP instead of DIG labeled dNTP was used as control. Quality of the DIG labeled probe was assessed by 

running a small amount of the probe on 1% agarose DNA gel (it always ran slower than control PCR 

product). The probe was denatured before use by incubating the probe at 100oC for 5 min, placing on ice 

for 2 min. For the exact position of probes in the DNA insert, see Figure 43.

Figure 43. DNA Insert Probes

AGP AGP

AGP ASN ASN GBS AGP PHL PHL GBS

GBS GBS fASN1 fPPO5 spacer fPPO5 fASN1 pPHL pR1 spacer pR1 pPHL

RV RV Hd RV RI RV RV Hd Sc Hd Sc

AGP2 AGP3 AGP2 AGP3

Standard probes

Additional probes

L B RB

RV=EcoRV, R1=EcoR1, Sc=Sca1, Hd=HindIII

LB = Left Border like region containing 25-bp Left Border and 162-bp flanking sequence.

RB = Right Border like region containing 25-bp Right Border and 161-bp flanking sequence.

Hybridization. The nylon membrane carrying transferred DNA was prehybridized in 40 ml pre-warmed 

DIG Easy Hybridization solution (Roche) at 42oC for 1-4 hrs in a bottle in a standard hybridization oven 

(Amerex Instruments Inc.) at 20-25 rpm. Hybridization was carried out by replacing the prehybridization 

buffer with a fresh amount of the same preheated solution, now containing 25-50 μl denatured DIG 

labeled probe, and continuing the incubation at 42oC, 20-25 rpm for about 16 hrs. The hybridization 

solution could be store at -20oC and reused up to 3 times. The reused hybridization solution was heated 

at 68oC for 10 minutes before use.

Detection. The hybridization solution was removed and replaced by 100 ml washing solution I (2X 

SSC/0.1% SDS). The membrane was washed twice in washing solution I for 10 min at room temperature. 

This low stringency buffer was poured off and preheated high stringency washing solution II (0.5X 

SSC/0.1% SDS, 600C) was added immediately. The membrane was washed twice in washing solution II at 

60-63oC for 20 min each at 25-30 rpm. This was followed by a brief rinse with 2X SSC to remove SDS. 

The membrane was rinsed with 150 ml of 1X DIG Washing Solution (Roche) in a tray for 2 min and 

incubated in 1X Blocking solution (Roche) for 0.5-3 hrs on a low-speed shaker. The blocked membrane 

was incubated with DIG antibody solution (1:10,000 dilution of Anti-DIG-alkaline phosphate conjugate 

with 1X Blocking solution) for 30 min on a shaker. The membrane was washed twice (15 min each) with 

1X DIG Washing Solution (Roche) and equilibrated with 1X detection buffer. The detection reaction was 

carried out with 2 ml CDP-Star solution (1:100 diluted stock of CDP-Star with 1X detection buffer) for 5 

min. The membrane was wrapped in a plastic film and exposed to the Z-ray film in the dark. Depending 

on the experiment, multiple exposures were taken from 30 sec to 30 min. The films were developed 

with Konica SRX-101A Z-ray film developer. The developed films were scanned to obtain the final 

images. 
J.R. Simplot Company Characterization of Inserted DNA 65

Explanation of Bands Visualized by DNA Gel Blot Analysis. DNA gel blot analysis was performed using 

DNA extracted from greenhouse-grown plants and digested with the following restriction enzymes: (1) 

EcoRV, (2) HindIII, and (3) EcoRI + ScaI. The positions of the restriction sites and probes employed are 

shown in Figure 43. The digested DNA was run with standard DIG-labeled molecular weight markers, 

DIGII and DIGVII, through 0.7% agarose gels in 0.5X TBE buffer and blotted onto Nylon Hybond N+ filters. 

The four DNA insert-derived probes used were AGP, PHL, ASN, and GBS (see Table 14). Additional 

probes used were AGP2 and AGP3. Each of the original films from hybridization experiments contained, 

apart from the 2 markers, three DNA digests of the untransformed control and the events for one 

variety. To facilitate interpretations, the original films were separated into three separate segments (one 

for each digest) with the marker lanes copied and boxed.

When reading the bands sizes in DNA gel blots, such observations are subject to interpretation by the 

reviewer and slight variation could occur between the expected and observed sizes. The observed sizes 

could be influenced by many factors including the sample matrix or slight differences in gel running 

conditions. Therefore, other information was considered, including the expected sizes and observations 

from other gels and digests when reporting the sizes of the bands. 

Table 14. Molecular Probes 

Probe

(bp)

Primer 

name

Primer sequence Genetic 

Elements

Position in 

pSIM1278

Size of hybridizing bands

EcoRV HindIII EcoRI-ScaI

AGP

(511)

AGP3F

AGP3R

CCAAAACTGAAAGTGGGAAACC

ACAACAACCCCCTCCTTGTG

AGP promoter 

fragment

17-527

4,247-4,757

OB: several

IB: 2,293

JB: 1,127+

OB: several

IB: 4230

JB: 2443+

OB: several

IB: 3751

JB: 3717+

ASN

(549)

HD076F2

HD076R2

GAACTCTTTATCCAGAAATG

TAGTCTCTATTGAATCTGCT

ASN1 and PPO5 

gene fragments

2,262-2,810

2,974-3,523

OB: several

IB: 712, 2293

JB: none

OB: several

IB: 4230

JB: none

OB: several

IB: none

JB: 3717+

PHL

(436)

PhoLF

PhoLR

AAAGTGTGAAGGGTATTTTTGTAAAC

CGAATGAGAGTGATAAGAGAGTGAGG

PHL promoter 

fragment

6,518-6,953

8,376-8,811

OB: several

IB: none

JB: 3567+

OB: several

IB: 1313

JB: 2163+

OB: several

IB: 3751

JB: 1903+

GBS

(546)

HD077F

HD077R

TGTGGTCTACAAAAAGGGGAAT

GGTAGCGGTAGGAGGGAGTT

Gbss promoter 

fragment

3,536-4,081

8,845-9,390

OB: several

IB: 2293

JB: 3567+

OB: several

IB: 4230

JB: 2163+

OB: several

IB: 3751

JB: 1903+

AGP2

(402)

HY1659

HY1660

ACAAGGAGGGGGTTGTTGTACT

CTTGATGTAAACACCGAATACGA

AGP promoter 

fragment

509-910

4,739-5,140

OB: several

IB: 2,293

JB: 1,127+

AGP3

(999)

HY1661

HY1662

TAAATTGGACGGACGATGAAAT

CGGAATTTACGAGAGGACCATA

AGP promoter 

fragment

1,130-2,128

5,360-6,358

OB: several

IB: 1, 225, 1,225

JB: none

PCR Confirmation of EcoR1/Scal Fragments in E, F, J, and H lines. The genomic DNA was isolated from 

the leaves of events E12, E24, F10, F37, J3, J55, J78, H37, and H50. The primers used for the 

confirmation of EcoR1/Scal fragments in E, F, J, and H lines were described in Figure 41. The PCR was 

carried out in 30 μl of PCR reaction mixture consisting of 3 μl of 10X PCR buffer, 0.6 μl of 10 mM dNTP, 

0.6 μl of 10 μM forward primer, 0.6 μl of 10 μM reverse primer, 100 ng of genomic DNA template, and 

0.2 μl of HotMaster Taq polymerase (Fisher BioReagents). The PCR amplification conditions were as 

follows: 1 cycle of 3 min at 95 °C followed by 40 cycles of 30 sec at 94 °C, 30 sec at 55°C, 2 min at 68 °C, 

and finishing with 10 min at 68 °C.
J.R. Simplot Company Characterization of Inserted DNA 66

CONCLUSIONS

Event Ranger Russet F10 contains a single copy of the DNA insert. As shown in Appendix 6. Field 

Performance and Tuber Evaluations and Appendix 9. Compositional Analyses, this insertion is not 

linked to any unexpected event-specific agronomic or biochemical changes. 

Event Ranger Russet F37 contains a single copy of the DNA insert. This insertion is not linked to any 

unexpected event-specific agronomic or biochemical changes.

Event Russet Burbank E12 contains a single copy of the DNA insert. This insertion is not linked to any 

unexpected event-specific agronomic or biochemical changes.

Event Russet Burbank E24 contains a single copy of the DNA insert with the exception of a 0.5-kb 

deletion at the Left Border side. This deletion does not affect construct efficacy. This insertion is not 

linked to any unexpected event-specific agronomic or biochemical changes.

Event Atlantic J3 contains one nearly intact copy of DNA insert with a small deletion at the Left Border 

site. Attached to this first copy is a second copy in a head-to-head orientation, whereby the second copy 

also had a small deletion at the Left Border site and a much larger deletion, comprising the R1/PhL

promoter silencing cassette, at the Right Border site. This insert arrangement resulted in a junction 

between part of the Agp promoter and flanking plant DNA that is unlikely to result in expression of a 

new open reading frame because the partial Agp promoter lacks the TATA-box, CCAAT-box, and 

additional 5’ elements needed to induce transcription. The insertions are not linked to any unexpected 

event-specific agronomic or biochemical changes.

Event Atlantic J55 contains one full copy of the DNA insert and an additional truncated copy lacking the 

R1/PhL promoter silencing cassette. The two copies integrated at the same site as an inverted repeat. 

The insertions are not linked to any unexpected event-specific agronomic or biochemical changes.

Event Atlantic J78 contains a single truncated DNA insert. The insert has a small deletion at the Left 

Border that did not affect efficacy of the associated Asn1/Ppo5 gene silencing cassette. A larger deletion

occurred at the Right Border site that included one of the promoters associated with the R1/PhL

promoter silencing cassette, and loss of efficacy of that cassette. This insertion is not linked to any 

unexpected event-specific agronomic or biochemical changes.

Event G G11 contains a single DNA insert insert with a large deletion at the Right Border site that 

includes the R1/PhL silencing cassette and results in a junction between the Agp promoter and flanking 

plant DNA. This flanking DNA does not contain known genes or new ORFs in the vicinity of the Agp

promoter (as deduced by BLAST analysis of a 2.5-kb flanking DNA fragment, see Appendix 4. Junction 

Analysis and Event-Specific Detection, indicating that it is unlikely for the insert to have altered the 

expression pattern of one or more plant genes. This insertion is not linked to any unexpected eventspecific agronomic or biochemical changes.

Event H H37 contains one complete DNA insert insert and three DNA insert fragments. One of the

inserted fragments consists of a small part of the Agp promoter of the DNA insert. This particular 

insertion may have resulted in the operable fusion of a promoter and open reading frame. However, if 

such a fusion did occur, it neither affected plant agronomics nor tuber biochemistry. The second 

truncated fragment lacks the Asn1/Ppo5 gene silencing cassette except for the Gbss promoter. The 
J.R. Simplot Company Characterization of Inserted DNA 67

remaining Gbss promoter is not functionally active because it lacks the 5’ sequences carrying CCAAT and 

TATA boxes. The fourth small fragment consists of a small part of the Agp promoter that is, for the same 

reasons, not functionally active. The insertions are not linked to any unexpected event-specific 

agronomic or biochemical changes.

Event H H50 contains one full copy of the DNA insert and an additional truncated copy lacking the 

R1/PhL promoter silencing cassette. The junction created by insertion of the truncated DNA insert

results in fusion of the Agp promoter with flanking plant DNA. It is possible that this fusion could result 

in the expression of an open reading frame. However, if such expression did occur, it neither affected 

plant agronomics nor tuber biochemistry. The insertions are not linked to any unexpected event-specific 

agronomic or biochemical changes.
J.R. Simplot Company Characterization of Inserted DNA 68

REFERENCES

Chawla R, Shakya R, Rommens CM (2012) Tuber specific silencing of asparagine synthetase-1 reduces 

the acrylamide-forming potential of potatoes grown in the field without affecting tuber shape 

and yield. Plant Biotechnol J, 10: 913-924.

Van Haaren MJJ, Sedee NJA, de Boer HA, Schilperoort RA, Hooykaas PJJ (1989) Mutational analysis of the 

conserved domains of a T-region border repeat of Agrobacterium tumefaciens. Plant Mol Bio, 

13: 523-531.