A key step in deregulating new transgenic traits is the molecular characterization of the transgene and insertion site. DNA LandMarks has developed unique expertise in this process and offer it as a service to our clients.

Event characterization includes:

• Determination copy number
• Confirmation of absence of vector backbone
• Sequencing of the transgene
• Sequencing of 500 – 1000 bp of flanking genomic sequence
• Confirms no ORFs were created or disrupted
• Demonstration inter-generational stability by high quality Southern blots

Use of Southern Blots is a key part of the transgenic characterization process:

• Samples digested with multiple enzymes
• Multiple probes are employed for hybridization
• Results determine:
• Copy number
• Backbone integration
• Insert integrity
• Approximate borderline of insert
• Additional Southern blotting can be done using lines from various generations – this will demonstrate intergenerational stability of the insert

Transgene Sequencing

• Transgene is cloned
• PCR products are sequenced three times to achieve Phred 40 sequence quality
• Sequences assembled and compared to original construct sequence
• DLM can perform analysis on multiple copy transformants if necessary

Flanking Sequence Recovery

• Transgene and flanking regions cloned and sequenced using Inverse PCR (IPCR)
• Genomic DNA is cut then ligated into plasmids
• These are sequenced and assembled to determine flanking region sequences
• DLM can perform analysis on multiple copy transformants if necessary