| Object : The aim of this project was to construct two level pools based three-dimension of Chinese fine wool merino sheep BAC library, at the same time, to design a high-throughput PCR rapid screening method to find out positive clones from 74,000 BACs, in order to offer mapping reagents and cloning materials to the physical mapping and map-based clonging on sheep genome.Method: Uesing the hierarchical pooling strategies, we constucted two levels pools with a three-dimensional pooling scheme of the BAC library of Chinese fine wool merino sheep. The primary row and column pools were based on the same row and column of every four 384-well microtiter plates, the primary plate pools were based on all clones of each 384-well microtiter plates. Then, we arranged the primary plate pools as 12 column×14 row, and constructed the secondary plate pools according the primary pooling strategies. Finally, we pooled the same labled primary row and column pools to the secondary row and column pool respectively. The method of sceening was PCR. It was one-step screening that we could screen a singal positive clone from 74,000 BACs, or three-step screening could screen more clones.The first step, we sceen the secondary plate, row and column pools to identitfy the candidate positive plate, row and column, at this step, a singal positive clone could be find out. More positive clones required another two steps.Result: We constructed 168, 672 and 1008 primary plate, row and column pools respectively, 26, 16 and 24 secondary plate, row and column pools respectively. By one-step screening(66 PCR reactions), we screened successfully a positive clone 373D13 with polymorphism marker BF94-1.Conclution: The two level pools we constructed could meet more than 300 PCR or hybridization sceening.The positive clones found out by our high-throughput PCR rapid screening method, could offer mapping reagents and cloning materials to the physical mapping and map-based clonging on sheep genome.
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