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The analysis of slippage synthesis of simple sequence DNA in vitro sheds some light on the question of how simple sequences arise in vivo. We show that it is possible to synthesize all types of repetitious di- and trinucleotide motifs starting from short primers and a polymerase in vitro. The rate of this synthesis depends on a sequence specific slippage rate, but is independent of the length of the fragments being synthesized. This indicates that only the ends of the DNA fragments are involved in determining this rate and that slippage is accordingly a short range effect. Slippage synthesis occurs also on a fixed template where only one strand is free to move, a situation which resembles chromosome replication in vivo. It seems therefore likely that slippage during replication is the cause of the observed length polymorphism of simple sequence stretches between individuals of a population.