BACKGROUND
in  <dig>   <dig>  million hectares of genetically modified organisms  have been planted in  <dig> countries  <cit> .
on the european union  market, the commercialization of gmos in the food/feed chain is subject to the eu legislation , which is becoming more and more complex to implement due to the increasing number and diversity of gmos  <cit> .
the majority of eu-authorized gmos  harbours the transgenic p35s element  35s promoter), the transgenic tnos element  or both of them, with an occurrence respectively reported of  <dig> ,  <dig>  and  <dig>  % .
to ensure the correct enforcement of the eu legislation, several gmo detection methods have been developed, mainly based on sybr®green and taqman® real-time pcr technologies.
usually, a screening is first performed with qpcr methods targeting the most common transgenic elements present in genetically modified  crops .
these strategies, covering a broad spectrum of gmos, allow to indicate the potential presence of gmos in tested samples .
in case of positive responses, eu-authorized gmos are subsequently identified and quantified using eu event-specific methods.
if some observed positive screening elements, like p35s and tnos, are not explained by these event-specific methods, the presence of eu-unauthorized gmos can be indirectly suspected  <cit> .
however, as most of the targeted elements originate from natural organisms , the confirmation of their presence can be irrefutably provided only by the characterization of the transgene flanking regions between the plant genome and the integrated cassette  <cit> .
to this end, dna walking strategies have notably been proposed in order to get this crucial information allowing to identify gm crops .
however, these metods are not usually used in gmo routine analysis because they are not easily implementable by the enforcement laboratories.
recently, an integrated dna walking strategy, better corresponding to the need of the enforcement laboratories, was developped to rapidly detect and identify eu-unauthorized gmos, without significant additional cost and equipment  <cit> .
this method targets the t35s element from the pcambia vector, which is frequent  in transgenic plants and is absent in eu-authorized gmos.
this dna walking approach, based on pcr, has the advantage to be fully integrated into the initial qpcr analysis as the same primers are used for the qpcr screening  and the dna walking   <cit> .
in addition, this approach was assessed as highly sensitive and able to deal with rice based mixtures and processed products, which is essential in gmo routine analysis  <cit> .
here, the concept of this integrated pcr-based dna walking strategy has been adapted to also target p35s and tnos, the most common transgenic elements found in gmos, in order to characterize a broader spectrum of gmos as well as to strengthen the initial dna walking system targeting t35s from pcambia.
for each element, two dna walking directions, starting from a position anchored on the sequences used for the p35s or tnos sybr®green qpcr screening, have been established  <cit> .
first, the p35s and tnos bidirectional dna walking methods were developed on bt rice, as previously used for the t35s pcambia method.
second, these dna walking methods were assessed using the certified reference material  of the gm maize mon <dig> , which represents a more complex matrix due to its large genome and its low target content.
finally, in order to illustrate its applicability in routine analysis, a gemma scheme proficiency test food matrix was submitted to the entire integrated strategy, including the qpcr screening using the p35s, tnos and t35s pcambia markers to detect the presence of gmos and, then, the dna walking methods, corresponding to the qpcr positive responses, allowing to characterize them.
RESULTS
in order to characterize a broad spectrum of both eu authorized and unauthorized gmos, two novel dna walking methods, based on the p35s and tnos transgenic elements, have been developed.
these methods were designed similarly to the t35s pcambia dna walking method targeting only eu unauthorized gmos  <cit> .
in the interest to provide an integrated approach, for each dna walking method, the same primers allow the detection of the potential presence of gmos containing the targeted elements  as well as their characterization and identification insofar as possible .
in silico study
since the dna walking approach is integrated into the screening step, the sybr®green primers published by barbau-piednoir et al.,  <dig> were used to target the p35s and tnos elements  <cit> .
as three primers are required by the dna walking method for each targeted element, an additional primer  intermediate to the screening primers  was designed .
the specificity of these primers was successfully assessed in silico, against all eu-authorized gmos, llps  and corresponding wts , using the software wemboss   <cit> .
moreover, for each of the targets, two walking directions were established  in order to extend the gmo coverage of the integrated dna walking strategy.table  <dig> oligonucleotide primers used for the real-time pcr assays, the dna walking approaches and the pcr confirmation of the transgenic junctions
development of the dna walking methods
assessment of p35s dna walking methods
for the p35s approach, several amplicons were observed from 100 % bt rice, corresponding to  <dig>  <dig> hges , for the four different degenerated random tagging  primers , including  <dig> amplicons for the p35s-f dna walking method  and  <dig> amplicons for the p35s-r dna walking method  .
the size range of these amplicons was approximately from 100 bp to  <dig> kbp and from  <dig> pb to  <dig> kbp for the p35s-f and p35s-r dna walking method, respectively.
all these amplicons were consecutively analysed by sequencing to evaluate the specificity of the methods .fig.
<dig> development of the bidirectional p35s and tnos dna walking methods on 100 % bt rice.
a visualisation of the obtained amplicons, numeroted from  <dig> to  <dig>  using the p35s and tnos dna walking methods applied on 100 ng of 100 % bt rice and wt rice.
for each method, four different drt primer mixes  have been used.
b for each dna walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the bt rice, is illustatred by the black arrows.
below the transgenic cassette, the sequence covering of the obtained amplicons from the 100 % bt rice is schematically represented by rectangles.
the corresponding amplicon numbering is indicated in the fig.
1a.
lb ; t35s ; hpt ; p35s ; lacz ; pubi ; cry1b ; tnos ; rb ; rice
all these characterized sequences corresponded specifically to the position of the p35s element in the transgenic cassette   <cit> .
as expected, these sequences present the continuity of the p35s element  for the p35s-f dna walking method  and the p35s promoter  regulating the hygromycin resistance gene   for the p35s-r dna walking method  .
for the wt rice sample, few amplicons  were observed  and identified as corresponding to the rice genome .
they are probably due to the use of drt primers which can potentially generate a background of aspecific products, especially in absence or in low amounts of targeted sequences  <cit> .
assessment of tnos dna walking methods
the use of the tnos dna walking approach with the two walking directions using the four different drt primers  on the 100 % bt rice sample produces several amplicons, including  <dig> amplicons for the tnos-f dna walking method  and  <dig> amplicons for the tnos-r dna walking method  .
the tnos-f and tnos-r dna walking methods gave respectively amplicons with a size range of approximately 100 bp to  <dig>  kbp and  <dig> pb to  <dig> kbp.
to assess the specificity of the methods, all these pcr products were examined by sequencing .
on the one hand, as expected, regarding the tnos element localisation in the transgenic cassette, 100 % of the analysed amplicons coming from the tnos-f dna walking method have allowed to characterize the transgene flanking regions between the rice genome and the right border of the integrated pcambia cassette via the amplicon sequences containing both the tnos element and the rice genome .
none of the obtained amplicons presented an unexpected sequence.
as the bt rice presents two transgenic insertions, two types of transgene flanking regions were characterized: one localised between the transgenic cassette  and a genomic sequence from chromosome ii of oryza sativa japonica group  identified using the amplicons generated by the drt c primers  and one situated between the pcambia cassette  and a genomic sequence from chromosome iii of oryza sativa japonica group  identified using the amplification coming from the drt a, b and d primers   <cit> .
these results yet clearly demonstrate the importance to use four different drt primer mixes.
indeed, the difference in affinity of these drt primers allows increasing the likelihood to successfully characterize all targets  <cit> .
in addition, the right border of the pcambia cassette on chromosome ii was shorter of two base-pairs compared to the one on chromosome iii .
these two transgene flanking regions were also properly confirmed by sequencing of pcr products obtained in using primers annealing to the pcambia cassette and chromosome ii or iii .
on the other hand, as expected, all pcr products generated from the tnos-r dna walking method allow to characterize the continuity of the tnos element  as well as, for the longer ones, the flanking region between the tnos element  and the cry1b gene  conferring an insect resistance  .
100 % of the analysed amplicons corresponded to the expected sequences.
similarly to the p35s dna walking methods, the bidirectional tnos approach presents uniquely specific amplifications further to the analysis of the bt rice sample while few aspecific amplicons , corresponding to the rice genome, were generated from the wt rice material  .
practical application of the dna walking methods
analysis of gm maize
to test the developed p35s and tnos bidirectional dna walking methods on a more complex food matrix than rice in term of genome size and target amount, gm maize mon <dig>  <dig>  % , corresponding to  <dig>  <dig> hges, was selected as it possesses both the p35s and tnos elements in its transgenic cassette  <cit> .
first, the presence of these elements in the tested crm sample was confirmed by sybr®green qpcr screening .
then, several amplicons were generated by each dna walking method with a size ranging from approximately 200 bp to  <dig> kbp .
in order to obtain the most informative sequences, the amplicon with the highest size for each drt primer mix in each applied dna walking method was selected to be sequenced .fig.
<dig> application of the bidirectional p35s and tnos dna walking methods on gm maize matrices.
a visualisation of the obtained amplicons using the p35s and tnos dna walking methods applied on 100 ng of the gm mon <dig> maize crm .
for each method, four different drt primer mixes  have been used.
the analyzed amplicons are indicated by a numerotation going from  <dig> to  <dig>  b for each dna walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the gm maize mon <dig>  is illustatred by the black arrows.
below the transgenic cassette, the sequence covering of the selected amplicons from the gm mon <dig> maize crm  and the gemma proficiency test food matrix  is schematically represented by rectangles.
the corresponding amplicon numbering is indicated in the fig.
2a and additional file  <dig>  lb ; p35s ; nptii ; tnos ; p4-as <dig> ; wtcab ; ract ; cry3bb <dig> ; tahsp <dig> ; rb ; maize
most of the selected amplicons from the p35s-f dna walking method present the 5’ transgene flanking region between the maize genome  and the p35s promoter  from the transgenic cassette of mon <dig>  as previously published   <cit> .
this transgene flanking region, confirming the presence of gm maize mon <dig>  is also targeted by the eu event-specific qpcr method to identify and quantify this gmo  <cit> .
only one tested amplicon  showed an aspecific sequence corresponding to the wt maize genome  .
a possible explanation is that the tested sample contains primarily wt maize material and only a relative low amount of the target.
for the p35s-r dna walking method, two different types of sequences were observed due to the presence of two p35s promoters in the transgenic cassette of gm mon <dig> maize   <cit> .
on the one hand, the continuity of the p35s promoter  regulated the neomycin phosphotransferase gene  from a. tumefaciens  which confers a resistance to kanamycin .
on the other hand, a part of the p35s promoter , followed by the 5’ upstream sequence of the wheat major chlorophyll a/b binding protein gene   and by the rice actin intron  , regulated the synthetic cry3bb <dig> gene , providing insect resistance .
the information acquired from these both types of sequences allows a better characterization of the transgenic cassette.
using the tnos-f dna walking method, all analysed amplicons showed a part of the tnos terminator from a. tumefaciens  followed by a modified p35s promoter, including in upstream of four repeats of a short activating sequence , referred to as 4-as <dig> promoter   <cit> .
this modified p35s promoter was then followed by the 5’ upstream sequence of the wheat major chlorophyll a/b binding protein gene   and the rice actin intron   to regulate the cry3bb <dig> gene  .
the tnos-r dna walking method provided the sequence of the nptii selection marker  followed by the tnos terminator  .
an aspecific sequence corresponding to the wt maize genome , identical to the amplicon n ° <dig> from the p35s-f dna walking method, was observed for the amplicons n°  <dig> and  <dig> .
most of the tested amplicons  derived from all the dna walking methods presented a sequence corresponding to the gm targets.
based on these data, the presence of gm mon <dig> maize in the tested sample was clearly identified by isolation and sequencing of its junction between the maize genome and the transgenic cassette.
in addition, this strategy allows to reconstruct  <dig>  kbp of the integrated transgenic cassette, going from the left border to a part of the gene cry3bb <dig>  in agreement with the published information   <cit> .
these results also highlight that the proposed dna walking strategy is able to identify gmos from different plant species.
analysis of the food matrix
in order to illustrate its applicability in gmo routine analysis by the enforcement laboratories, the entire workflow of the integrated system was applied on a food matrix  from a gemma scheme proficiency test containing  <dig>  % of gm maize mon <dig> event, corresponding to  <dig> hges.
first, similarly to the gmo routine analysis, the gemma food matrix was submitted to the sybr®green qpcr screening using the p35s, tnos and t35s pcambia screening markers allowing to detect the potential presence of gmos .
as expected, a positive signal was observed for the p35s and tnos screening markers while the t35s pcambia screening marker gave a negative signal , suggesting the potential presence of gmos in the tested food matrix.
second, based on the positive signals obtained from the screening qpcr analysis, the bidirectional p35s and tnos dna walking approaches were selected to be applied on the sample.
in doing so, the potential presence of gmos will be confirmed by the characterization of their sequences.
all applied dna walking methods were able to produce amplicons in a size range from approximately 200 bp to  <dig>  kbp .
in order to follow an efficient workflow suitable for gmo routine analysis, only one amplicon, chose for its large size as well as for its ease to be selected on an electrophoresis gel, was sequenced for each dna walking method .
with all these dna walking methods, 100 % of the analysed amplicons presented sequences specific to the gm target.
indeed, when using p35s-f dna walking, the transgene flanking region between the maize genome  and the p35s promoter from the transgenic cassette of mon <dig>  was identified, proving the presence of this gmo in the tested sample  <cit> .
the p35s-r dna walking method presented the continuity of the p35s promoter  regulating the nptii selection marker .
from the tnos-f dna walking method, a part of the tnos terminator , followed respectively by the 4as- <dig> promoter  and the wheat major chlorophyll a/b binding protein gene  , was detected.
via the tnos-r dna walking method, the sequence of the nptii gene  regulated by the tnos terminator  was observed .
all these sequences indubitably prove the presence of the gm mon <dig> maize event in the gemma food matrix sample though the identification of its junction between the maize genome and the transgenic cassette as well as the partial reconstruction of its transgenic cassette, in agreement with the published information  <cit> .
similarly to the t35s pcambia dna walking method, the good specificity of the newly developed dna walking methods  was illustrated in this study since almost all of the sequences from the analysed amplicons generated from the bt rice , mon863- <dig>  %  and gemma proficiency test  matrices corresponded to the gm targets  <cit> .
the success of this strategy is mainly due to the specificity of the target-specific primers, allowing to initially amplify the targets by pcr and, then, to enrich them by two successive semi-nested pcrs .
CONCLUSIONS
in order to provide an integrated system able to detect, characterize and identify a broad spectrum of both eu authorized and unauthorized gmos in food/feed matrices, two bidirectional dna walking methods targeting p35s or tnos, the most common transgenic elements, were developed to be anchored on the sequences used for the p35s or tnos qpcr sybr®green screening described by barbau-piednoir et al.,  <dig>  <cit> .
these dna walking methods also allow to strengthen the previously published t35s pcambia dna walking method in order to currently target around 75 % of the gm crops  <cit> , personal communication.
first, the p35s and tnos bidirectional dna walking methods were developed and assessed for their specificity using 100 % bt rice.
these methods were evaluated as highly specific since no aspecific amplifications were generated in presence of the target.
second, the developed dna walking methods were tested on a more complex maize food matrix, in term of genome size, containing approximately 10 % of the gm maize mon <dig> event.
finally, the entire workflow of the integrated system, including the detection of the potential presence of gmos by qpcr screening with the p35s, tnos and t35s pcambia markers and, subsequently, the confirmation of their presence using the dna walking methods corresponding to the previously obtained qpcr responses, was applied on a gemma scheme proficiency test matrix, containing  <dig>  % of the gm maize mon <dig> event, to illustrate its applicability in gmo routine analysis by the enforcement laboratories.
for all tested matrices, the p35s and the tnos bidirectional dna walking methods were successfully applied as the gmo presence was proven via the characterization of the junction between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette.
in addition to its clear benefit in gmo detection, this integrated system has the advantage to present a simple procedure and a short time-frame to get the results.
however, in order to analyse even more easily the pcr products derived from the dna walking methods, some adaptations in the entire dna walking workflow could be done regarding the purification of the generated amplicons excised from the electrophoresis gel and the subsequent sequencing using sanger technology.
indeed, even if the initial dna walking workflow remains simple, in case of matrices containing several gmos, harbouring the same targeted element, the purification of the potential numerous amplicons excised from the electrophoresis gel and the subsequent sanger sequencing could be cumbersome.
this situation could be for instance encountered with matrices presenting a low amount of eu-unauthorized gmos mixed with eu-authorized gmos harbouring the elements p35s and/or tnos, very frequently observed in gm crops.
in this scenario, the obtained amplicons will present different sequences, representing potentially one gmo per observed dna fragment.
therefore, the simplified workflow, consisting in selecting the largest size amplicons to obtain the most informative sequences, does not guarantee the entire representativeness of gmos present in the tested sample.
consequently, it’s preferable to analyse all amplicons observed on the electrophoresis gel and to eventually them using sanger technology, which may be a quite laborious work.
in the future, this difficulty could be circumvented in replacing the step related to the purification of the amplicons excised from the electrophoresis gel and the subsequent sanger sequencing by a high-throughput next-generation-sequencing approach, as suggested by liang et al.,  <dig>  <cit> .

 in million hectares of genetically modified organisms have been planted in countries. on the european union market, the commercialization of gmos in the food/feed chain is subject to the eu legislation, which is becoming more and more complex to implement due to the increasing number and diversity of gmos. the majority of eu-authorized gmos harbours the transgenic p35s element 35s promoter), the transgenic tnos element or both of them, with an occurrence respectively reported of, and  %. to ensure the correct enforcement of the eu legislation, several gmo detection methods have been developed, mainly based on sybr®green and taqman® real-time pcr technologies. usually, a screening is first performed with qpcr methods targeting the most common transgenic elements present in genetically modified crops. these strategies, covering a broad spectrum of gmos, allow to indicate the potential presence of gmos in tested samples. in case of positive responses, eu-authorized gmos are subsequently identified and quantified using eu event-specific methods. if some observed positive screening elements, like p35s and tnos, are not explained by these event-specific methods, the presence of eu-unauthorized gmos can be indirectly suspected. however, as most of the targeted elements originate from natural organisms, the confirmation of their presence can be irrefutably provided only by the characterization of the transgene flanking regions between the plant genome and the integrated cassette. to this end, dna walking strategies have notably been proposed in order to get this crucial information allowing to identify gm crops. however, these metods are not usually used in gmo routine analysis because they are not easily implementable by the enforcement laboratories. recently, an integrated dna walking strategy, better corresponding to the need of the enforcement laboratories, was developped to rapidly detect and identify eu-unauthorized gmos, without significant additional cost and equipment. this method targets the t35s element from the pcambia vector, which is frequent in transgenic plants and is absent in eu-authorized gmos. this dna walking approach, based on pcr, has the advantage to be fully integrated into the initial qpcr analysis as the same primers are used for the qpcr screening and the dna walking. in addition, this approach was assessed as highly sensitive and able to deal with rice based mixtures and processed products, which is essential in gmo routine analysis. here, the concept of this integrated pcr-based dna walking strategy has been adapted to also target p35s and tnos, the most common transgenic elements found in gmos, in order to characterize a broader spectrum of gmos as well as to strengthen the initial dna walking system targeting t35s from pcambia. for each element, two dna walking directions, starting from a position anchored on the sequences used for the p35s or tnos sybr®green qpcr screening, have been established. first, the p35s and tnos bidirectional dna walking methods were developed on bt rice, as previously used for the t35s pcambia method. second, these dna walking methods were assessed using the certified reference material of the gm maize mon, which represents a more complex matrix due to its large genome and its low target content. finally, in order to illustrate its applicability in routine analysis, a gemma scheme proficiency test food matrix was submitted to the entire integrated strategy, including the qpcr screening using the p35s, tnos and t35s pcambia markers to detect the presence of gmos and, then, the dna walking methods, corresponding to the qpcr positive responses, allowing to characterize them.  in order to characterize a broad spectrum of both eu authorized and unauthorized gmos, two novel dna walking methods, based on the p35s and tnos transgenic elements, have been developed. these methods were designed similarly to the t35s pcambia dna walking method targeting only eu unauthorized gmos. in the interest to provide an integrated approach, for each dna walking method, the same primers allow the detection of the potential presence of gmos containing the targeted elements as well as their characterization and identification insofar as possible. in silico study since the dna walking approach is integrated into the screening step, the sybr®green primers published by barbau-piednoir et al., were used to target the p35s and tnos elements. as three primers are required by the dna walking method for each targeted element, an additional primer intermediate to the screening primers was designed. the specificity of these primers was successfully assessed in silico, against all eu-authorized gmos, llps and corresponding wts, using the software wemboss. moreover, for each of the targets, two walking directions were established in order to extend the gmo coverage of the integrated dna walking strategy.table oligonucleotide primers used for the real-time pcr assays, the dna walking approaches and the pcr confirmation of the transgenic junctions development of the dna walking methods assessment of p35s dna walking methods for the p35s approach, several amplicons were observed from 100 % bt rice, corresponding to hges, for the four different degenerated random tagging primers, including amplicons for the p35s-f dna walking method and amplicons for the p35s-r dna walking method. the size range of these amplicons was approximately from 100 bp to kbp and from pb to kbp for the p35s-f and p35s-r dna walking method, respectively. all these amplicons were consecutively analysed by sequencing to evaluate the specificity of the methods.fig. development of the bidirectional p35s and tnos dna walking methods on 100 % bt rice. a visualisation of the obtained amplicons, numeroted from to using the p35s and tnos dna walking methods applied on 100 ng of 100 % bt rice and wt rice. for each method, four different drt primer mixes have been used. b for each dna walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the bt rice, is illustatred by the black arrows. below the transgenic cassette, the sequence covering of the obtained amplicons from the 100 % bt rice is schematically represented by rectangles. the corresponding amplicon numbering is indicated in the fig. 1a. lb; t35s; hpt; p35s; lacz; pubi; cry1b; tnos; rb; rice all these characterized sequences corresponded specifically to the position of the p35s element in the transgenic cassette. as expected, these sequences present the continuity of the p35s element for the p35s-f dna walking method and the p35s promoter regulating the hygromycin resistance gene for the p35s-r dna walking method. for the wt rice sample, few amplicons were observed and identified as corresponding to the rice genome. they are probably due to the use of drt primers which can potentially generate a background of aspecific products, especially in absence or in low amounts of targeted sequences. assessment of tnos dna walking methods the use of the tnos dna walking approach with the two walking directions using the four different drt primers on the 100 % bt rice sample produces several amplicons, including amplicons for the tnos-f dna walking method and amplicons for the tnos-r dna walking method. the tnos-f and tnos-r dna walking methods gave respectively amplicons with a size range of approximately 100 bp to kbp and pb to kbp. to assess the specificity of the methods, all these pcr products were examined by sequencing. on the one hand, as expected, regarding the tnos element localisation in the transgenic cassette, 100 % of the analysed amplicons coming from the tnos-f dna walking method have allowed to characterize the transgene flanking regions between the rice genome and the right border of the integrated pcambia cassette via the amplicon sequences containing both the tnos element and the rice genome. none of the obtained amplicons presented an unexpected sequence. as the bt rice presents two transgenic insertions, two types of transgene flanking regions were characterized: one localised between the transgenic cassette and a genomic sequence from chromosome ii of oryza sativa japonica group identified using the amplicons generated by the drt c primers and one situated between the pcambia cassette and a genomic sequence from chromosome iii of oryza sativa japonica group identified using the amplification coming from the drt a, b and d primers. these results yet clearly demonstrate the importance to use four different drt primer mixes. indeed, the difference in affinity of these drt primers allows increasing the likelihood to successfully characterize all targets. in addition, the right border of the pcambia cassette on chromosome ii was shorter of two base-pairs compared to the one on chromosome iii. these two transgene flanking regions were also properly confirmed by sequencing of pcr products obtained in using primers annealing to the pcambia cassette and chromosome ii or iii. on the other hand, as expected, all pcr products generated from the tnos-r dna walking method allow to characterize the continuity of the tnos element as well as, for the longer ones, the flanking region between the tnos element and the cry1b gene conferring an insect resistance. 100 % of the analysed amplicons corresponded to the expected sequences. similarly to the p35s dna walking methods, the bidirectional tnos approach presents uniquely specific amplifications further to the analysis of the bt rice sample while few aspecific amplicons, corresponding to the rice genome, were generated from the wt rice material. practical application of the dna walking methods analysis of gm maize to test the developed p35s and tnos bidirectional dna walking methods on a more complex food matrix than rice in term of genome size and target amount, gm maize mon  %, corresponding to hges, was selected as it possesses both the p35s and tnos elements in its transgenic cassette. first, the presence of these elements in the tested crm sample was confirmed by sybr®green qpcr screening. then, several amplicons were generated by each dna walking method with a size ranging from approximately 200 bp to kbp. in order to obtain the most informative sequences, the amplicon with the highest size for each drt primer mix in each applied dna walking method was selected to be sequenced.fig. application of the bidirectional p35s and tnos dna walking methods on gm maize matrices. a visualisation of the obtained amplicons using the p35s and tnos dna walking methods applied on 100 ng of the gm mon maize crm. for each method, four different drt primer mixes have been used. the analyzed amplicons are indicated by a numerotation going from to b for each dna walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the gm maize mon is illustatred by the black arrows. below the transgenic cassette, the sequence covering of the selected amplicons from the gm mon maize crm and the gemma proficiency test food matrix is schematically represented by rectangles. the corresponding amplicon numbering is indicated in the fig. 2a and additional file lb; p35s; nptii; tnos; p4-as; wtcab; ract; cry3bb; tahsp; rb; maize most of the selected amplicons from the p35s-f dna walking method present the 5’ transgene flanking region between the maize genome and the p35s promoter from the transgenic cassette of mon as previously published. this transgene flanking region, confirming the presence of gm maize mon is also targeted by the eu event-specific qpcr method to identify and quantify this gmo. only one tested amplicon showed an aspecific sequence corresponding to the wt maize genome. a possible explanation is that the tested sample contains primarily wt maize material and only a relative low amount of the target. for the p35s-r dna walking method, two different types of sequences were observed due to the presence of two p35s promoters in the transgenic cassette of gm mon maize. on the one hand, the continuity of the p35s promoter regulated the neomycin phosphotransferase gene from a. tumefaciens which confers a resistance to kanamycin. on the other hand, a part of the p35s promoter, followed by the 5’ upstream sequence of the wheat major chlorophyll a/b binding protein gene and by the rice actin intron, regulated the synthetic cry3bb gene, providing insect resistance. the information acquired from these both types of sequences allows a better characterization of the transgenic cassette. using the tnos-f dna walking method, all analysed amplicons showed a part of the tnos terminator from a. tumefaciens followed by a modified p35s promoter, including in upstream of four repeats of a short activating sequence, referred to as 4-as promoter. this modified p35s promoter was then followed by the 5’ upstream sequence of the wheat major chlorophyll a/b binding protein gene and the rice actin intron to regulate the cry3bb gene. the tnos-r dna walking method provided the sequence of the nptii selection marker followed by the tnos terminator. an aspecific sequence corresponding to the wt maize genome, identical to the amplicon n ° from the p35s-f dna walking method, was observed for the amplicons n° and. most of the tested amplicons derived from all the dna walking methods presented a sequence corresponding to the gm targets. based on these data, the presence of gm mon maize in the tested sample was clearly identified by isolation and sequencing of its junction between the maize genome and the transgenic cassette. in addition, this strategy allows to reconstruct kbp of the integrated transgenic cassette, going from the left border to a part of the gene cry3bb in agreement with the published information. these results also highlight that the proposed dna walking strategy is able to identify gmos from different plant species. analysis of the food matrix in order to illustrate its applicability in gmo routine analysis by the enforcement laboratories, the entire workflow of the integrated system was applied on a food matrix from a gemma scheme proficiency test containing  % of gm maize mon event, corresponding to hges. first, similarly to the gmo routine analysis, the gemma food matrix was submitted to the sybr®green qpcr screening using the p35s, tnos and t35s pcambia screening markers allowing to detect the potential presence of gmos. as expected, a positive signal was observed for the p35s and tnos screening markers while the t35s pcambia screening marker gave a negative signal, suggesting the potential presence of gmos in the tested food matrix. second, based on the positive signals obtained from the screening qpcr analysis, the bidirectional p35s and tnos dna walking approaches were selected to be applied on the sample. in doing so, the potential presence of gmos will be confirmed by the characterization of their sequences. all applied dna walking methods were able to produce amplicons in a size range from approximately 200 bp to kbp. in order to follow an efficient workflow suitable for gmo routine analysis, only one amplicon, chose for its large size as well as for its ease to be selected on an electrophoresis gel, was sequenced for each dna walking method. with all these dna walking methods, 100 % of the analysed amplicons presented sequences specific to the gm target. indeed, when using p35s-f dna walking, the transgene flanking region between the maize genome and the p35s promoter from the transgenic cassette of mon was identified, proving the presence of this gmo in the tested sample. the p35s-r dna walking method presented the continuity of the p35s promoter regulating the nptii selection marker. from the tnos-f dna walking method, a part of the tnos terminator, followed respectively by the 4as- promoter and the wheat major chlorophyll a/b binding protein gene, was detected. via the tnos-r dna walking method, the sequence of the nptii gene regulated by the tnos terminator was observed. all these sequences indubitably prove the presence of the gm mon maize event in the gemma food matrix sample though the identification of its junction between the maize genome and the transgenic cassette as well as the partial reconstruction of its transgenic cassette, in agreement with the published information. similarly to the t35s pcambia dna walking method, the good specificity of the newly developed dna walking methods was illustrated in this study since almost all of the sequences from the analysed amplicons generated from the bt rice, mon863-  % and gemma proficiency test matrices corresponded to the gm targets. the success of this strategy is mainly due to the specificity of the target-specific primers, allowing to initially amplify the targets by pcr and, then, to enrich them by two successive semi-nested pcrs.  in order to provide an integrated system able to detect, characterize and identify a broad spectrum of both eu authorized and unauthorized gmos in food/feed matrices, two bidirectional dna walking methods targeting p35s or tnos, the most common transgenic elements, were developed to be anchored on the sequences used for the p35s or tnos qpcr sybr®green screening described by barbau-piednoir et al.,. these dna walking methods also allow to strengthen the previously published t35s pcambia dna walking method in order to currently target around 75 % of the gm crops, personal communication. first, the p35s and tnos bidirectional dna walking methods were developed and assessed for their specificity using 100 % bt rice. these methods were evaluated as highly specific since no aspecific amplifications were generated in presence of the target. second, the developed dna walking methods were tested on a more complex maize food matrix, in term of genome size, containing approximately 10 % of the gm maize mon event. finally, the entire workflow of the integrated system, including the detection of the potential presence of gmos by qpcr screening with the p35s, tnos and t35s pcambia markers and, subsequently, the confirmation of their presence using the dna walking methods corresponding to the previously obtained qpcr responses, was applied on a gemma scheme proficiency test matrix, containing  % of the gm maize mon event, to illustrate its applicability in gmo routine analysis by the enforcement laboratories. for all tested matrices, the p35s and the tnos bidirectional dna walking methods were successfully applied as the gmo presence was proven via the characterization of the junction between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette. in addition to its clear benefit in gmo detection, this integrated system has the advantage to present a simple procedure and a short time-frame to get the results. however, in order to analyse even more easily the pcr products derived from the dna walking methods, some adaptations in the entire dna walking workflow could be done regarding the purification of the generated amplicons excised from the electrophoresis gel and the subsequent sequencing using sanger technology. indeed, even if the initial dna walking workflow remains simple, in case of matrices containing several gmos, harbouring the same targeted element, the purification of the potential numerous amplicons excised from the electrophoresis gel and the subsequent sanger sequencing could be cumbersome. this situation could be for instance encountered with matrices presenting a low amount of eu-unauthorized gmos mixed with eu-authorized gmos harbouring the elements p35s and/or tnos, very frequently observed in gm crops. in this scenario, the obtained amplicons will present different sequences, representing potentially one gmo per observed dna fragment. therefore, the simplified workflow, consisting in selecting the largest size amplicons to obtain the most informative sequences, does not guarantee the entire representativeness of gmos present in the tested sample. consequently, it’s preferable to analyse all amplicons observed on the electrophoresis gel and to eventually them using sanger technology, which may be a quite laborious work. in the future, this difficulty could be circumvented in replacing the step related to the purification of the amplicons excised from the electrophoresis gel and the subsequent sanger sequencing by a high-throughput next-generation-sequencing approach, as suggested by liang et al.,. 
in order to provide a system fully integrated with qpcr screening, usually used in gmo routine analysis, as well as being able to detect, characterize and identify a broad spectrum of gmos in food/feed matrices, two bidirectional dna walking methods targeting p35s or tnos, the most common transgenic elements found in gm crops, were developed. finally, to illustrate its applicability in gmo routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qpcr screening to detect the potential presence of gmos and the subsequent dna walking methods to characterize and identify the detected gmos, was applied on a gemma scheme proficiency test matrix. due to their simple procedure and their short time-frame to get results, the developed dna walking methods proposed here can be easily implemented in gmo routine analysis by the enforcement laboratories. second, the methods were assessed on a maize sample containing a low amount of the gm mon event, representing a more complex matrix in terms of genome size and sensitivity. in providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this dna walking strategy is a key molecular tool to prove the presence of gmos in any given food/feed matrix. first, the newly developed dna walking methods, anchored on the sequences used for the p35s or tnos qpcr screening, were tested on bt rice that contains these two transgenic elements. via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of gmos was properly confirmed or infirmed in all tested samples.
 in order to provide a system fully integrated with qpcr screening, usually used in gmo routine analysis, as well as being able to detect, characterize and identify a broad spectrum of gmos in food/feed matrices, two bidirectional dna walking methods targeting p35s or tnos, the most common transgenic elements found in gm crops, were developed. these newly developed dna walking methods are completing the previously implemented dna walking method targeting the t35s pcambia element. methods food/feed matrices containing transgenic crops were analysed using the integrated dna walking system.  first, the newly developed dna walking methods, anchored on the sequences used for the p35s or tnos qpcr screening, were tested on bt rice that contains these two transgenic elements. second, the methods were assessed on a maize sample containing a low amount of the gm mon event, representing a more complex matrix in terms of genome size and sensitivity. finally, to illustrate its applicability in gmo routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qpcr screening to detect the potential presence of gmos and the subsequent dna walking methods to characterize and identify the detected gmos, was applied on a gemma scheme proficiency test matrix. via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of gmos was properly confirmed or infirmed in all tested samples.  due to their simple procedure and their short time-frame to get results, the developed dna walking methods proposed here can be easily implemented in gmo routine analysis by the enforcement laboratories. in providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this dna walking strategy is a key molecular tool to prove the presence of gmos in any given food/feed matrix. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 