Thymidine kinase deficient (TK-) cells are killed on HAT medium, which contains the drug aminopterin. Aminopterin blocks the endogenous pathway of nucleotide production by inhibiting the enzyme dihydrofolate reductase (DHFR), which catalyzes the first reaction in the utilization of dihydrofolate in nucleotide biosynthesis.

Further, the TK- cells are unable to utilize the thymidine present in the medium for nucleotide production. As a result, TK- cells die of nucleotide, starvation on HAT medium. Therefore, thymidine kinase gene can be used as a selectable marker only when TK- host cells are used for transfection.

The transfected cells are cultured on the HAT medium on which only TK+ cell can survive and multiply. The requirement of TK- host cells is a serious limitation of this marker. Therefore, selectable markers with more general applicability to non-mutant animal cell lines have been developed; such markers are often called dominant selectable markers.