Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

S.J. Zheng, G. Henken, E. Sofiari, E. Jacobsen, F.A. Krens

Research output: Contribution to journalArticleAcademicpeer-review

23 Citations (Scopus)

Abstract

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration
Original languageEnglish
Pages (from-to)237-245
JournalTransgenic Research
Volume10
Issue number3
DOIs
Publication statusPublished - 2001

Fingerprint

Shallots
shallots
Onions
Allium cepa
DNA Sequence Analysis
Ligation
genetically modified organisms
genomics
Polymerase Chain Reaction
DNA
Genome
hybridization
Genetically Modified Plants
Plant DNA
Genetic Transformation
T-DNA
Agrobacterium tumefaciens
genome
Organism Cloning

Keywords

  • polymerase chain-reaction
  • mediated transformation
  • arabidopsis-thaliana
  • plasmid rescue
  • amplification
  • walking
  • plants
  • gene
  • junctions
  • fragments

Cite this

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title = "Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders",
abstract = "Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration",
keywords = "polymerase chain-reaction, mediated transformation, arabidopsis-thaliana, plasmid rescue, amplification, walking, plants, gene, junctions, fragments",
author = "S.J. Zheng and G. Henken and E. Sofiari and E. Jacobsen and F.A. Krens",
year = "2001",
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language = "English",
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Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders. / Zheng, S.J.; Henken, G.; Sofiari, E.; Jacobsen, E.; Krens, F.A.

In: Transgenic Research, Vol. 10, No. 3, 2001, p. 237-245.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

AU - Zheng, S.J.

AU - Henken, G.

AU - Sofiari, E.

AU - Jacobsen, E.

AU - Krens, F.A.

PY - 2001

Y1 - 2001

N2 - Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration

AB - Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration

KW - polymerase chain-reaction

KW - mediated transformation

KW - arabidopsis-thaliana

KW - plasmid rescue

KW - amplification

KW - walking

KW - plants

KW - gene

KW - junctions

KW - fragments

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DO - 10.1023/A:1016633410041

M3 - Article

VL - 10

SP - 237

EP - 245

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

IS - 3

ER -