Scholarlink

<p>Rapid detection of inserted transgenes and their genomic functions is essential for molecular characterization of Living Modified Organisms (LMOs). The <i>Phalaenopsis</i> event 311 NR harbors a T-DNA cassette comprising the CaMV 35S promoter, TMV Omega, hygromycin phosphotransferase, the <i>Commelina communis F3′5′H</i> gene, the nos, and CaMV terminators, resulting in blue-purple flowers consistent with altered anthocyanin biosynthesis. Universal PCR screening confirmed the presence of CaMV 35S promoter (142&#xa0;bp) and nos terminator (194&#xa0;bp) fragments. To enhance detection specificity, four primer pairs each for CaMV 35S promoter and nos terminator were designed and validated on Gateway entry vectors, with 60&#xa0;°C identified as the optimal annealing temperature. Application to genomic DNA yielded junction-informative amplicons of approximately 3&#xa0;kb and 1&#xa0;kb. Comparative sequencing across platforms demonstrated complementary capabilities. Illumina provided high accuracy and coverage suitable for detecting conserved elements, while PacBio enabled structural resolution of insertion contexts. Nanopore sequencing, combined with inverse PCR enrichment, concentrated junction-bearing molecules and facilitated the recovery of transgenic elements spanning both insert and flanking genomic regions. Informative contigs across platforms preserved the conserved order of regulatory and marker elements, supporting consistent structural interpretation. These results establish iPCR-Nanopore as a cost-efficient and field-deployable approach for rapid detection and junction localization, while Illumina and PacBio remain indispensable for complete locus reconstruction. Together, the three platforms support a tiered workflow in which targeted detection is followed by comprehensive structural resolution, thereby advancing molecular monitoring of LMOs in ornamental plants.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Rapid detection of transgene insertion patterns in living modified Phalaenopsis via inverse PCR and nanopore sequencing validated against Illumina and PacBio sequencing platforms

  • 期刊论文

  • 发表日期：

    2025年10月24日

查看全文
  • Yu Kyong Hu,
  • Jun-Kyung Lee,
  • Yong-Gil Shin,
  • Byoung-Eun Min,
  • Ki‐Hong Jung,
  • Chan Hui Lee,
  • Sun-Goo Hwang,
  • Sung Don Lim

摘要

Rapid detection of inserted transgenes and their genomic functions is essential for molecular characterization of Living Modified Organisms (LMOs). The Phalaenopsis event 311 NR harbors a T-DNA cassette comprising the CaMV 35S promoter, TMV Omega, hygromycin phosphotransferase, the Commelina communis F3′5′H gene, the nos, and CaMV terminators, resulting in blue-purple flowers consistent with altered anthocyanin biosynthesis. Universal PCR screening confirmed the presence of CaMV 35S promoter (142 bp) and nos terminator (194 bp) fragments. To enhance detection specificity, four primer pairs each for CaMV 35S promoter and nos terminator were designed and validated on Gateway entry vectors, with 60 °C identified as the optimal annealing temperature. Application to genomic DNA yielded junction-informative amplicons of approximately 3 kb and 1 kb. Comparative sequencing across platforms demonstrated complementary capabilities. Illumina provided high accuracy and coverage suitable for detecting conserved elements, while PacBio enabled structural resolution of insertion contexts. Nanopore sequencing, combined with inverse PCR enrichment, concentrated junction-bearing molecules and facilitated the recovery of transgenic elements spanning both insert and flanking genomic regions. Informative contigs across platforms preserved the conserved order of regulatory and marker elements, supporting consistent structural interpretation. These results establish iPCR-Nanopore as a cost-efficient and field-deployable approach for rapid detection and junction localization, while Illumina and PacBio remain indispensable for complete locus reconstruction. Together, the three platforms support a tiered workflow in which targeted detection is followed by comprehensive structural resolution, thereby advancing molecular monitoring of LMOs in ornamental plants.