Become a major field of interest for the design of high-throughput methods for parallel expression of proteins in multiple expression systems. To serve rapid cloning, several technologies were established in recent years including e.g. the commercial Gateway and Creator recombination systems and the proprietary In-Fusion assembly technology likely based on the 39-59 exonuclease activity of poxvirus DNA polymerase generating complementary 15-bp overhangs between target and destination DNA molecules. Additionally, novel restriction enzyme/DNA ligase-mediated vector construction methods were established including BioBrick assembly and Golden Gate cloning. Ligation-independent cloning, sometimes also referred to as ligase-independent cloning, is a simple, rapid and relatively cheap method for the generation of expression constructs. It uses the 39-.59 exonuclease activity of T4 DNA polymerase to create specific single-stranded, 59-extending tails of nucleotides in DNA fragments and complementary single-stranded overhangs in the target vector. Compared with previous studies, the COV in our control group was somewhat larger. A possible explanation for this difference could be given by following two reasons. One is the relatively smaller number of subjects, and the other is the relatively larger variability in age because D2R subfamily in each extrastriatal region is known to show age-related decline. In vitro brain homogenate binding studies have demonstrated that D2R subfamily exists in two affinity states, i.e., high and low affinity states. The high affinity state is thought to represent the functional state, and agonists bind preferentially to D2R subfamily in the high affinity state, while antagonists have equal affinity for D2R subfamily in the high and low affinity states. In vivo competition studies between endogenous dopamine and a labeled agonist or antagonist ligand estimated the percentage of high affinity state to be about 60–70%. On the other hand, some recent in vivo studies indicated that most D2R subfamily is in the high affinity state at living conditions because the binding of exogenous unlabeled agonist to D2R subfamily in high or low affinity states could not be differentiated with either a labeled agonist or antagonist ligand. Thus, the accurate proportion of the two states remains controversial. In this study, relatively low D2/D3 occupancy rates by pramipexole were mainly due to low dose of pramipexole. However, based on the two states theory, another reason may be because D2/D3 occupancy rates by agonist pramipexole were estimated by antagonist ligand 11C-FLB 457.