Intravesicular isothermal replication. Lanes 1 and 7 contain a 50 bp DNA ladder. Lanes 2 and 3 are from unencapsulated reactions and lanes 4-6 are within phospholipid vesicles. Lanes 2 and 3 are isothermal replication reactions under standard conditions except that 0.5 mM CaCl2 was added. CaCl2 is needed for proteinase K activity. Further, lane 3 contained 0.9 units of proteinase K, demonstrating that proteinase K is capable of digesting components of the tHDA system and thus inhibiting DNA amplification. A similar experiment was conducted inside of vesicles with proteinase K added to the outside of the vesicles (lane 4) and proteinase K added to both the inside and the outside of the vesicles (lane 5). Finally, to further demonstrate that the reaction was encapsulated, dNTPs were added extravesicularly resulting in an inability to replicate DNA since dNTPs are incapable of crossing POPC membranes (lane 6).