To develop a PCR based multiplex assay that can be used to authenticate Chinese hamster ovary cell lines which are important in the production of biological therapeutics.
- Identify tetranucleotide STR markers by searching the CHO genome using BLAST
- Design and optimize a functional multiplex PCR assay
- Determine specificity of the assay
- Determine stability of the STR markers over high passage number
Tetranucleotide short tandem repeat (STR) markers were identified in the CHO-K1 genome using a bioinformatics approach (BLAST). Primers were designed flanking the STR target regions and were screened using PCR and gel electrophoresis. Forward primers were labeled with a fluorescent dye and further screening of primer sets were completed using capillary electrophoresis. Multiplex reactions were tested and optimized (varying the concentrations of DNA, primers, etc.). Allele distributions were determined by testing CHO DNA from several sources.