Identification of a heteromorphic microsatellite within the thymidine kinase gene in L5178Y mouse lymphoma cells

Abstract

The objective of this work is to identify a heteromorphism within the thymidine kinase (Tk1) gene which can be used to assay for allele loss by means of PCR. Intron F of mouse Tk1 contains two (CA)_n microsatellite sequences separated by 107 bp of non-repetitive sequence. We tested this region for heteromorphism in L5178Y mouse lymphoma cells. A PCR primer pair designated Ag11 yielded products of 396 and 194 bp from L5178Y tk^+/− genomic DNA. The 194-bp product resulted from a secondary binding site between the two (CA)_n repeats for the forward Ag11 primer and was not produced from tk^−/− mutants that had lost the functional Tk1_b allele. Ag12 primers produced two PCR products of 523 and ∼440 bp and Ag13 primers produced products of 579 and ∼ 500 bp. In both these cases, the difference in product size was approximately equal, indicating that Intron F is ∼ 80 bp shorter in the non-functional Tk1_a allele than in Tk1_b. This heteromorphism forms the basis for an assay for allele loss by means of PCR. Ag11 and Ag13 primers yielded additional products of 91 and 274 bp, respectively, consistent with sizes expected from the mouse Tk1 pseudogenes (Tk1-ps). Our conclusions drawn from an analysis of 122 mutants for Tk1_b loss using Ag12 primers agreed with previous analysis of the NcoI heteromorphism. Thus, a simple PCR-based analysis can identify Tk1_b loss in the L5178Y mouse lymphoma cells.