J.R. Simplot Company Junction Analysis and Event-Specific Detection 1

Appendix 4

Junction Analysis and Event-Specific Detection

INDEX

Section Page

Index 1

Introduction 2

Results 3

Materials and Methods 29

Conclusions 31

References 33

Certification 34
J.R. Simplot Company Junction Analysis and Event-Specific Detection 2

INTRODUCTION

At least one DNA insert/flanking plant DNA junction was sequenced using either Adapter LigationMediated PCR or Thermal Asymmetric Interlaced PCR. The junction sequence was used to design 

primers for each event, and these primers were applied for event-specific PCR-based detection 

methods. The specificity of the primers was tested on all the events submitted for deregulation to 

ensure that there was no cross-reactivity between the detection systems. The methods developed may

be used to monitor plants and tubers in field and storage as part of quality management programs.
J.R. Simplot Company Junction Analysis and Event-Specific Detection 3

RESULTS

Ranger Russet Event F10

Based on the DNA insert organization of F10 (see Figure 1 below and Appendix 1. Characterization of 

Inserted DNA), a fragment was amplified at the Right Border site. Sequence analysis determined that 

the DNA insert lacked the first 31 bp upstream from the Right Border cleavage site.

Figure 1. F10 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).

The primers used for line-specific PCR were F10 1F, which is derived from the DNA insert, and F10 1R 

from the flanking plant DNA (Figure 1). The amplified DNA fragment for the line-specific PCR had a size 

of 1076 bp (Figure 2).

The single DNA insert of F10 had a 31 bp deletion at the Right Border side that did not affect the 

functional activity of the adjacent silencing cassette.
J.R. Simplot Company Junction Analysis and Event-Specific Detection 4

Figure 2. Ranger Russet F10: Line-Specific PCR Using Primer Pair F10 1F and F10 1R

1kb = 1 kb DNA marker lane (Invitrogen), 100 bp = 100 bp DNA marker lane (invitrogen), FC = Ranger 

Russet control (untransformed), NC = negative control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 5

Ranger Russet Event F37

Based on the DNA insert organization of F37 (see Figure 3 below and Appendix 1. Characterization of 

Inserted DNA), a fragment was amplified at the Right Border site. The junction fragment consists of a 

truncated Right Border side of the DNA insert (which means that the integrated DNA insert lacks the 

Right Border and a small adjacent upstream sequence of 13 bp) and is followed by, first, a 13 bp plantderived ‘stuffer’ DNA and, second, a small fragment (33 bp) of the DNA insert that represents an internal 

sequence of the trailer of the Ppo gene that is present in the silencing cassette of pSIM1278.

Figure 3. F37 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).

Two primers, one from the DNA insert and another from the sequenced flanking DNA, F37AF and F37AR

(Figure 3), were used to amplify a 1091 bp F37-specific junction fragment (Figure 4).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 6

Figure 4. Ranger Russet F37: Line-Specific PCR Using Primer Pair F37 AF and F37 AR. Amplified F37-

specific fragment at Right Border side.

1kb = 1 kb DNA marker lane (Invitrogen), 100 bp = 100 bp DNA marker lane (invitrogen), FC = Ranger 

Russet control (untransformed), NC = negative control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 7

Russet Burbank Event E12

The DNA insert organization of E12 is shown in Figure 5 below. A 547 bp fragment at the Right Border 

site lacked the first 92 bp upstream from the Right Border cleavage site. A 609 bp fragment at the Left 

Border site lacked 17 bp downstream from the Left Border cleavage site (Figure 6A and B).

Figure 5. E12 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 8

Figure 6A. Russet Burbank E12: Line-Specific PCR. Amplified E12-specific fragment at the Right Border 

side, using primers HY1599 and HY1600.

 

1kb = 1 kb DNA marker lane (Invitrogen), 100 bp = 100 bp DNA marker lane (invitrogen), EC = Russet

Burbank control (untransformed), NC = negative control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 9

Figure 6B. Russet Burbank E12: Line-Specific PCR. Amplified E12-specific fragment at Left Border side 

using primer HY1617 and HY1620.

1kb = 1 kb DNA marker lane (Invitrogen), 100 bp = 100 bp DNA marker lane (invitrogen), EC = Russet

Burbank control (untransformed), NC = negative control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 10

Russet Burbank Event E24

TAIL-PCR was used to isolate DNA insert junction fragments from event E24 using the nested, Right 

Border side-specific primers HY1557, HY1558, HY1576 and HY1577 (Table 1). Based on the differential 

shift between the second and third products on agarose gels, E24-specific PCR products were extracted 

and cloned. Sequence analysis of the junction fragment demonstrates that E24 carries a full-length DNA 

insert (up to the cleavage site within the Right Border). Two primers, one annealing to a sequence within 

the DNA insert (HY1599) and one annealing to a site in the adjacent plant DNA (HY1601) (see Appendix 

1. Characterization of Inserted DNA, Figure 7 shown below), were used to amplify a 543 bp E24-specific 

DNA fragment (Figure 8).

Figure 7. Insert for E24

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).

Sequence analysis demonstrated that the primers used for line-specific PCR are the DNA insert specific 

primer HY1599 and HY1601 which is from flanking DNA-derived DNA (Figure 7).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 11

Figure 8. Russet Burbank E24: Line-Specific PCR Using Primer Pair HY1599 and HY1601. Amplified E24-

specific fragment at Right Border side.

1kb = 1 kb DNA marker lane (Invitrogen), 100 bp = 100 bp DNA marker lane (invitrogen), EC = Russet

Burbank control (untransformed), NC = negative control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 12

Atlantic Event J3

Event J3 contains one nearly intact and one partial copy of the DNA insert, connected head to head at 

the Left Border site, with deletions of Left Border regions and parts of the adjacent Agp promoters (see 

Appendix 1. Characterization of Inserted DNA and Figure 9 below).

Figure 9. J3 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).

Different combinations of primers at the ends of the inserts were used to amplify DNA fragments, which 

were then analyzed by sequencing. It was found that one copy had a 430 bp deletion at the Left Border 

side while the other copy showed a 251 bp deletion at the Left Border side. The resulting structure 

comprises an inverted repeat carrying two truncated Agp promoters whereby the longer promoter 

fragment contains a 179 bp sequence that functions as spacer. To develop a line-specific PCR method, 

we designed one primer, HY1768, that anneals to an Agp promoter sequence on the one side of the 

connection and a second primer, HY1772, annealing to the Agp on the other side (Figure 9). As shown in 

Figure 10, these primers could be used to amplify a line specific 464 bp DNA fragment from J3 DNA but 

not from DNA of untransformed Atlantic, nor events J55 or J78.
J.R. Simplot Company Junction Analysis and Event-Specific Detection 13

Figure 10. Atlantic J3: Line-Specific PCR Using Primer Pair HY1768 and HY1772. Amplified J3-specific 

fragment between two DNA inserts at Left Border sides.

100 bp = 100 bp DNA marker lane (invitrogen), JC = Atlantic control (untransformed), NC = negative 

control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 14

Atlantic Event J55

Both Southern analyses and TAIL PCR indicated that event J55 had two connected copies of the DNA 

insert. The Right Border region of the first DNA insert is connected to a truncated internal Agp region of 

the second DNA insert in the reverse orientation (see Figure 11 below and Appendix 1. Characterization 

of Inserted DNA).

Figure 11. J55 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).

A short plant-derived 49 bp stuffer sequence is present between the two DNA inserts. The J55-specific 

forward primer JY981 was designed to anneal to the right side of the Gbss promoter of the first DNA 

insert and was 29-nt away from the left side of RB. The J55-specific reverse primer JY982 was targeted 

to the Agp promoter of the second DNA insert and was 187 nucleotides away from the right side of RB 

(Figure 11). As shown in Figure 12, a line specific 401 bp DNA fragment was amplified with these primers 

in event J55 but neither in untransformed Atlantic nor in J3 or J78.
J.R. Simplot Company Junction Analysis and Event-Specific Detection 15

Figure 12. Atlantic J55: Line-Specific PCR Using Primer Pair JY981 and JY982. Amplified J55-specific 

fragment.

100 bp = 100 bp DNA marker lane (invitrogen), JC = Atlantic control (untransformed), NC = negative 

control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 16

Atlantic Event J78

Based on the truncated structure of the DNA insert of J78 (see Figure 13 below and Appendix 1. 

Characterization of Inserted DNA), a fragment of DNA flanking the Right Border side was sequenced. 

This sequence was used to design a primer, J78R, that, together with the DNA insert primer J78F (Figure 

13) was used to amplify a J78-specific DNA fragment.

Figure 13. J78 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).

As shown in Figure 14, the 513 bp DNA fragment amplified with primers J78F and J78R was specific for

J78. Sequencing the junction demonstrated that the DNA insert had a 1583 bp deletion at the Right 

Border side.
J.R. Simplot Company Junction Analysis and Event-Specific Detection 17

Figure 14. Atlantic J78: Line-Specific PCR Using Primer Pair J78F and J78R. Amplified J78-specific 

fragment at Right Border side.

100 bp = 100 bp DNA marker lane (invitrogen), JC = Atlantic control (untransformed), NC = negative 

control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 18

G event G11

Based on the truncated structure of the DNA insert of G11 (see Figure 15 below and Appendix 1. 

Characterization of Inserted DNA), the sequence of DNA flanking the Right Border side was determined. 

Two primers, one annealing to a sequence within the DNA insert (HY1680) and the other one annealing 

to flanking plant DNA (HY1683) (Figure 15), were used to amplify a 734 bp G11-specifc junction 

fragment (Figure 16). Sequence analysis of the fragment demonstrated that the DNA insert lacked 3,317

bp at the Right Border side, which means that it contained a full Asn1/Ppo5 gene silencing cassette and 

lacked the R1/PhL promoter silencing cassette.

Figure 15. Insert for G11

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 19

Figure 16. G11: Line-Specific PCR Using Primer Pair HY1680 and HY1683. Amplified G11-specific 

fragment (734 bp) at Right Border side. 

100 bp = 100 bp DNA marker lane (invitrogen), GC = G control variety (untransformed), G9 = G event, 

not included in this submission, NC = negative control PCR reaction (without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 20

H event H37

Based on the DNA insert organization of H37 (see Figure 17 below and Appendix 1. Characterization of 

Inserted DNA), a Right Border junction fragment was isolated using the nested primers (HY1557, 

HY1558, and HY1576) by TAIL-PCR. Sequencing of the fragment made it possible to design two primers, 

one annealing to a sequence within the DNA insert (JY939) and the other one annealing to a flanking 

plant DNA sequence (JY938) (Figure 17).

Figure 17. H37 Insert

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).

The event-specific primers were used to amplify the junction. As demonstrated in Figure 18, the 553 bp 

DNA fragment was amplified with H37 DNA but not with DNA from either the conventional variety H or 

the event H50. 
J.R. Simplot Company Junction Analysis and Event-Specific Detection 21

Figure 18. H37: Line-Specific PCR Using Primer Pair JY938 and JY939. Amplified H37-specific fragment 

(553 bp) at Right Border side. 

100 bp = 100 bp ladder (Invitrogen), HC = H Control, H74 = H event not included in this submission, NC = 

negative control (PCR reaction without plant DNA).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 22

H event H50

The DNA insert organization of H50 is shown in Figure 19 below and Appendix 1. Characterization of

Inserted DNA. A junction fragment was isolated using TAIL-PCR with the nested primers HY1562, 

HY1566, and HY1567 (Table 2).

Figure 19. Insert for H50

Arrows below the diagram (→) indicate the approximate position of primers used to amplify an eventspecific DNA fragment. Promoter orientation indicated by arrows ( ).

Based on the size difference between the first and second PCR products on agarose gels, specific PCR 

products were cloned and sequenced. The H50 Left Border junction specific primers JY979 and JY980 

were used to amplify a line specific 507-bp junction fragment in the H50 event but not in the 

conventional variety H nor H37 (Figure 20). 
J.R. Simplot Company Junction Analysis and Event-Specific Detection 23

Figure 20. H50: Line-Specific PCR Using Primer Pair JY979 and JY980. Amplified H50-specific fragment 

at Left Border side.

M = 100 bp ladder (Invitrogen), HC = H Control, H74 = event not included in this submission, NC = 

negative control (PCR reaction without plant DNA).

 
J.R. Simplot Company Junction Analysis and Event-Specific Detection 24

Line-Specificity of PCR-Based Detection Methods.

As shown in Figure 21, the 10 event-specific PCR methods do not cross react with the other events in 

this deregulation submission.

Figure 21. Line-Specific PCR Detection Methods

 

M = 100-bp ladder from invitrogen; EWT = Russet Burbank Control; FWT = Ranger Russet Control; JWT = 

Atlantic Control; GWT = G Control; HWT = H Control. NC = negative control (PCR reaction without plant 

DNA). Test lines as indicated in the text (F10, F37).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 25

Figure 21 (Continued). Line-Specific PCR Detection Methods

M = 100 bp ladder from invitrogen; EWT = Russet Burbank Control; FWT = Ranger Russet Control; JWT = 

Atlantic Control; GWT = G Control; HWT = H Control. NC = negative control (PCR reaction without plant 

DNA). Test lines as indicated in the text (E12, E24).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 26

Figure 21 (Continued). Line-Specific PCR Detection Methods

M = 100 bp ladder from invitrogen; EWT = Russet Burbank Control; FWT = Ranger Russet Control; JWT = 

Atlantic Control; GWT = G Control; HWT = H Control. NC = negative control (PCR reaction without plant 

DNA). Test lines as indicated in the text (J3, J55).

 
J.R. Simplot Company Junction Analysis and Event-Specific Detection 27

Figure 21 (Continued). Line-Specific PCR Detection Methods

M = 100 bp ladder from invitrogen; EWT = Russet Burbank Control; FWT = Ranger Russet Control; JWT = 

Atlantic Control; GWT = G Control; HWT = H Control. NC = negative control (PCR reaction without plant 

DNA). Test lines as indicated in the text (J78, G11).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 28

Figure 21 (Continued). Line-Specific PCR Detection Methods

M = 

100bp ladder from invitrogen; EWT = Russet Burbank Control; FWT = Ranger Russet Control; JWT = 

Atlantic Control; GWT = G Control; HWT = H Control. NC = negative control (PCR reaction without plant 

DNA). Test lines as indicated in the text (H37, H50).
J.R. Simplot Company Junction Analysis and Event-Specific Detection 29

MATERIALS AND METHODS

Potato material. Leaves from greenhouse-grown events and untransformed plants were used to extract 

DNA and isolate and characterize the DNA insert junctions.

Adapter ligation-mediated PCR Junction fragments were amplified by PCR using digested DNA ligated 

with adapter primers AP1 and AP2 as described by O’Malley et al. (2007) and detailed below. The 

amplified product containing a DNA insert primer binding site was selected by a secondary PCR reaction. 

The sequences of DNA insert primers used to analyze RB and LB junctions are shown in Tables 1 and 2, 

respectively. Plant DNA was isolated using either the CTAB method or the Qiagen Plant DNAeasy kit: 

200-500 ng DNA was digested for 3-5 hours with a restriction enzyme for which an adapter had been 

designed (EcoRI, HindIII, BamHI, AseI/NdeI) in a total volume of 20 μl. The digested DNA (3 μl) was 

ligated with its respective adapter (1 μl) in a mixture containing 0.5 μl T4 DNA ligase, 0.3 μl ATP, 1.0 μl 

10X ligation buffer, and 4.2 μl water. The primary PCR was carried out using the first DNA insert specific 

primer and AP1 with Hot Master Taq polymerase (Fisher BioReagents) under the following amplification 

conditions: 1 cycle of 3 min at 95°C followed by 30 cycles of 30 sec at 94°C, 30 sec at 60°C, 2.5 min at 

68°C, and finishing with 10 min at 68°C. A 1 μl aliquot of the primary PCR product was used for 

secondary reactions ( 1 cycle of 3 min at 95°C; 35 cycles of 30 sec at 94°C, 30 sec at 62°C, 2.5 min at 

68°C; 1 cycle of 10 min at 68°C) with the second DNA insert specific primer and AP2. This protocol was 

performed at the same time for non-transformed control plants and the events. Products of the 

secondary PCR were run on 1% agarose in TAE buffer. Bands unique for the events were gel-extracted 

using a Qiagen QIAquick Gel Extraction kit, cloned into pGEM-T Easy vector (Promega, Madison WI), and 

sequenced. Primers specific for DNA insert-flanking DNA were used, together with a DNA insert primer, 

to amplify junction fragments. 

Table 1. DNA Insert Primers Used to Analyze RB Junction Fragments

Primer Name Primer position in pSIM1278

PHL-GBSS 8822- 8844

GBSS-PR 9756- 9777

HY1669 1675 - 1698 ; 5905- 5928

HY1670 6479- 6504

HY1671 6516- 6541

HY1557 8824- 8848

HY1558 4011- 4035; 9320- 9344

HY1576 9751- 9777

HY1577 9846- 9869

RB3R-T1a 7696- 7719

RB3L-T1b 7736- 7759

RB3R-T1d 7866- 7889
J.R. Simplot Company Junction Analysis and Event-Specific Detection 30

Table 2. DNA Insert Primers Used to Analyze LB Junction Fragments

Primer Name Primer position in pSIM1278

HY1562 460- 485; 4690- 4715 (reverse)

HY1566 19643-7 (reverse)

HY1567 19590- 19614 (reverse)

Thermal asymmetric interlaced (TAIL) PCR. This method was used as an alternative to adapter ligationmediated PCR. Three series of reactions using AD (Arbitrary Degenerate) and SP (nested sequencespecific) primers were carried out to obtain DNA insert junction fragments. The standard 20 μl reaction 

mixture consisted of 2 μl 10X PCR buffer, 0.4 μl 10 mM dNTP, 0.4 μl 10 μM SP1, 5 μl 8-16 μM AD, 1 μl 

template, with either 0.1 μl HotMaster Taq polymerase (extension temperature 68 °C) or TaKaRa Ex 

TaqTM (TAKARA BIO Inc). A 1 μl aliquot of 50ng/μl leaf DNA was used as template for the first PCR; 1 μl of 

a 1:50 dilution of the first reaction was used as template for the second PCR; and 1 μl of 1:50 dilution of 

the second reaction was used for the third PCR. The second and third reactions were run on a gel, and 

bands with sizes that changed between the second and third reactions were cloned into pGEM-T Easy 

vector (Promega, Madison WI) and sequenced. Primers specific for DNA insert-flanking DNA were used, 

together with DNA insert-specific primers to clone junction fragments that were sequenced to confirm 

their identity. 
J.R. Simplot Company Junction Analysis and Event-Specific Detection 31

CONCLUSIONS

At least one junction where the DNA insert meets the flanking plant DNA was sequenced for each of the 

10 events and those sequences were used to design event-specific PCR-based detection methods (see 

summary of primers for each event in Table 3). The specificity of the primers was tested on all the 

events to ensure that there was no cross-reactivity with other events. The methods may be used to 

monitor plants and tubers in field and storage as part of quality management programs and can be 

developed further to provide for routine testing in food.

Table 3. Primers Used for Event-specific PCR

Event Ranger Russet F10: Primers F101F and F101R can be used to amplify an F10-specific DNA 

fragment of 1076 bp, and thus results in a line specific test method for event F10.

Event Ranger Russet F37: Primers F37AF and F37AR can be used to amplify an F37-specific DNA 

fragment of 1091 bp, and thus results in a line specific test method for event F37.

Event Russet Burbank E12: Primers HY1617 and HY1620 can be used to amplify an E12-specific DNA 

fragment of 609-bp at the Left Border, and primers HY1599 and HY1600 can be used to amplify a 547 bp 

fragment at the Right Border, and thus either set of primers could result in a line specific test method 

for event E12.

Event Russet Burbank E24: Primers HY1599 and HY1601 can be used to amplify an E24-specific DNA 

fragment of 543 bp, and thus results in a line specific test method for event E24.

Event Atlantic J3: Primers HY1768 and HY1772 can be used to amplify a J3-specific DNA fragment of 464

bp, and thus results in a line specific test method for event J3.

Event Atlantic J55: Primers JY981 and JY982 can be used to amplify a J55-specific DNA fragment of 401

bp, and thus results in a line specific test method for event J55.

Event Forward Primer Reverse Primer PCR Product (bp)

F10 F101F F101R 1076

F37 F37AF F37AR 1091

E12 (Left Border) HY1617 HY1620 609

E12 (Right Border) HY1599 HY1600 547

E24 HY1599 HY1601 543

J3 HY1768 HY1772 464

J55 JY981 JY982 401

J78 J78F J78R 513

G11 HY1680 HY1683 734

H37 JY939 JY938 553

H50 JY979 JY980 507
J.R. Simplot Company Junction Analysis and Event-Specific Detection 32

Event Atlantic J78: Primers J78F and J78R can be used to amplify a J78-specific DNA fragment of 513 bp, 

and thus results in a line specific test method for event J78.

Event G11: Primers HY1680 and HY1683 can be used to amplify a G11-specific DNA fragment of 734 bp, 

and thus provide an event-specific test method for G11.

Event H37: Primers JY938 and JY939 can be used to amplify an H37-specific DNA fragment of 553 bp, 

and thus provide an event-specific test method for event H37.

Event H50: Primers JY979 and JY980 can be used to amplify an H50-specific DNA fragment of 507 bp, 

and thus provide an event-specific test method for event H50.
J.R. Simplot Company Junction Analysis and Event-Specific Detection 33

REFERENCES

O’Malley RC, Alonso JM, Kim CJ, Leisse TJ, Ecker JR (2007) An adapter ligation-mediated PCR method for 

high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nature Protocols, 2:2910-2917.