The aim of this proposal is to screen a minimum of 5,000 STSs in 450 individuals per year. To achieve this throughput, we will screen upfront a plate of 90 ethnically diverse individuals. Only in the case when a frequent polymorphism is seen, or several different DHPLC profiles are observed, indicating the presence of various polymorphisms, will we analyze the remaining 360 samples individually. However, if no or only a polymorphism of low frequency is observed, we will pool at least four chromosomes to achieve adequate throughput. Based on present experience, only every fourth or fifth STS will require individual analysis of all individuals. In order to maximize sample throughput on the DHPLC systems, we will tag amplicons with different fluorophores. Present technology allows simultaneous detection of at least two such tags without spectral overlap. Further increases in throughput are planned by means of mass spec tags. For this purpose, STSs that can be analyzed at the same temperature as determined by our DHPLC melting algorithm will be labeled with different mass spec tags. Following their amplification, denaturation and reannealing, amplicons will be combined and separated on a single DHPLC column. Following resolution of hetero- and homoduplex molecules, the separated species will pass through a photolysis unit, in which the mass spec tags are cleaved off. Only the mass spec tags will then be subjected to analysis in a quadrupole mass spectrometer to identify the eluted peaks. Since different mismatches have been shown to yield different profiles, only a few representative samples, an expected average of 5 samples per STS screened, need to be sequenced in order to establish the chemical nature of a mismatch.
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