Stability Assays
Nucleic Acid Sequencing Services
BioReliance provides a comprehensive DNA sequencing service in accordance with ICH, FDA and CHMP guidelines on genetic stability. Nucleic acid analysis can be used to verify the coding sequence of the protein and physical state of the expression construct. BioReliance has experience in the sequencing of plasmids, vectors, bacterial, mammalian and insect DNA. The nucleotides sequence is determined by dye terminator cycle sequencing using fully automated Applied Biosystems 3130xl 16 capillary Genetic Analyzer. These multiple capillary systems include software for complete analysis and data management.
Services include:
- Double stranded and single strand template sequencing
- PCR amplicon sequencing including mRNA transcripts for genetic stability
- Single primer extension
- Molecular cloning
- Flexible sequencing strategies and redundancy
- Database searches
FISH Services
Fluorescence in situ hybridization (FISH) allows the chromosomal location of recombinant DNA sequences inserted into a cell line to be determined. If more than one site is present, FISH can determine the relative distribution of the sequences between those sites. In parallel with copy number determination and DNA sequencing, the technique allows the stability of the inserted DNA sequences to be confirmed. Assays are designed individually, depending on the inserted plasmid or plasmids and the host cell line.
The technique can also be applied to map the chromosomal location of genes and to identify individual human and mouse chromosomes and sex chromosomes of various species, using commercial chromosome paints.
Genetic Stability Assays
BioReliance provides a full range of tests for the characterization and determination of genetic stability of expression constructs. These tests are performed in accordance with the relevant regulatory guidelines including FDA, EMEA and ICH. BioReliance has experience in testing both mammalian (for cell line characterization) and prokaryotic systems. Genetic stability testing includes analysis of chromosomally inserted expression systems or extrachromosomal systems and mRNA transcripts encoding the gene product. Testing includes copy number determinations of expression cassettes,
- Restriction endonuclease fragment mapping (examination for insertion/mutation/integration sites/rearrangements)
- Southern blotting
- Northern blotting
- DNA sequencing
- Fluorescent in situ hybridization
