Detection of single copy gene sequences from single trypanosomes
Detection of single copy gene sequences from single trypanosomes
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DOI：
10.1016/s0166-6851(96)02810-1
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发表时间：
1997-02-01
影响因子：
1.5
通讯作者：
Tait, A
中科院分区：
文献类型：
--
作者：
MacLeod, A;Turner, CMR;Tait, A
最初的实验使用稀释提取的相当于单个基因组（0.12 pg）的布鲁氏锥虫dna，每次反应表明，使用嵌套引物在技术上是可行的，将单个复制序列（TIM，三磷酸异构酶基因）在溴化乙啶染色凝胶上扩增到可见水平。为了确定是否有可能从单个锥虫中扩增TIM位点，从感染了stib386和TREU 927混合或单独感染TREU 927的小鼠中分离出血流形式。在单次TREU 927感染的16只锥虫中，有5只出现了预测大小的溴化乙锭染色带。在混合感染的19只单锥虫中，12只出现了80 bp （TREU 927）或100 bp （STIB 386）的溴化乙啶染色片段，没有出现2个片段。
Initial experiments using dilutions of extractedTrypanosoma bruceiDNA equivalent to a single genome (0.12 pg) per reaction showed that, using nested primers it was technically feasible to amplify a single copy sequence (TIM, triosephosphate isomerase gene) to visible levels on an ethidium bromide-stained gel. To determine whether it was possible to amplify the TIM locus from a single trypanosome, bloodstream forms were isolated from mice which had been infected with either a mixture of stocks STIB 386 and TREU 927, or with TREU 927 alone. Of 16 single trypanosomes from the single TREU 927 infections, 5 gave an ethidium bromide-stained band of the predicted size. Of 19 single trypanosomes from the mixed infections, 12 gave an ethidium bromide-stained fragment of either 80 bp (TREU 927) or 100 bp (STIB 386): none gave 2 fragments.