Genotyping with Microsatellites Assures Cell Line Identity and Culture Purity

The development of genotyping methods provides the Coriell Cell Repositories with the means to identify and track cell lines through all of the operations necessary to establish, expand, freeze, recover, and distribute them to outside laboratories.

Over the course of the past several years, CCR has established an extensive program of genotyping based on microsatellite polymorphisms. Six highly polymorphic microsatellites have a combined matching probability of one in 33,000,000 for unrelated individuals. The characteristics of each marker are provided in the following table:

Microsatellite Marker Range of Allele Sizes (bp) Heterozygosity pM (matching probability)
THO-1 154-178 0.77 0.086; 1 out of 12
D5S592 166-206 0.83 0.051; 1 out of 20
D10S526 182-266 0.84 0.017; 1 out of 59
vWA31 127-167 0.81 0.062; 1 out of 16
D22S417 172-213 0.85 0.039; 1 out of 25
FES/FPS 206-234 0.67 0.165; 1 out of 6

Alleles are identified by their fluorescence, and the size in bases of each allele is determined by its migration compared with co-electrophoresed size standards labeled with a different fluorochrome. Allele sizes are downloaded into the Repository database. All freeze recoveries and expansions of a cell line are genotyped, and the allele sizes are compared to those already recorded in the database. This procedure assures correct identity of the cell lines.

One great benefit of genotyping cell cultures is that contamination of one cell culture by another is readily detected by the presence of three or more alleles per microsatellite.

The CCR also offers a genotyping kit containing the reagents and the primers for the PCR amplification of five of the six the microsatellites. These primers are labeled with a combination of 6-FAM and HEX for sensitive detection in many fragment size analysis procedures.

The kit provides instructions for multiplexing PCR, all reagents, except for the thermostable DNA polymerase, and a control samples of known genotype for use in testing the procedure and for comparison of relative fragment sizes. This kit will provide investigators with a convenient and easy method to record and to confirm the identity of cell lines.