Multiplex Pcr Primers are picked from 100 bp from the ends of contig Primo and screened against the entire database vith a stringency of 12/20as a cutoff Primers synthesized on MerMade in 95-vell format (Average concentration is19 pmol/ul) One u l of each primer is pooled for each PCR reaction and the pool is dried and resuspended in 10 ul sdd Ho 9 C for 6 min nce nutes(once) Agarose gel 6 until analysis Cleanup with shrimp alkaline phosphatase "Extract with phenol/chloroform Precipitate vith 2.5 volumes of ethanol acetate and wash vitd n sdh, o in the original volume Tvo ul of the PCR product is thermocycled vith each of the primers individually according to the standard BigDye conditions Primers that generate sequences are removed from the pool and PCR is repeated 2.3.5着色互补 PCR (color comp l ementation assay) 荧光PCR的原理是用不同 的荧光染料,如红色的罗丹明 (6-carboxy-x-rhodamin, POX)I 绿色荧光素 (5-carboxyfluorescein, FAM)S 分别标记于不同的寡核苷酸引 物上,同时扩增多个DNA区段 反应完毕后,利用分子筛除去多 余的引物。用紫外线照射扩增产 物,就能显示某一种或几种荧光 染料颜色的组合,如果某一 DNA区带缺失,则会缺乏相应 的颜色,据此可以很快诊断是否 某种基因缺失,或是发现某些感2.3.5 着色互补 PCR(color complementation assay) 荧光 PCR 的原理是用不同 的荧光染料，如红色的罗丹明 (6-carboxy-x-rhodamin, POX)和 绿色荧光素 (5’-carboxyfluorescein，FAM)等 分别标记于不同的寡核苷酸引 物上，同时扩增多个 DNA 区段， 反应完毕后，利用分子筛除去多 余的引物。用紫外线照射扩增产 物，就能显示某一种或几种荧光 染料颜色的组合，如果某一 DNA 区带缺失，则会缺乏相应 的颜色，据此可以很快诊断是否 某种基因缺失，或是发现某些感