影响荧光终止法PCR循环测序反应的因素

Effects of concentrations of templates and primers on dye terminator cycle sequencing

比较了一些影响荧光终止法PCR循环测序反应的因素。实验结果显示在BeckmanCEQ2 0 0 0自动测序仪上 ,可读序列长度随着pUC18模板量增加而逐渐增多 ,当模板量达到 12 5ng时DNA可读序列最长 ,以后随着pUC18量增加测序长度逐渐下降。当引物量是 1μl时 ,其测序结果比用 0 .5μl引物时好。在同样模板量情况下 ,10 μl反应体积比 5

Some parameters such as the amount of template and the amount of primer in Dye Terminator Cycle Sequencing (DTCS) are discussed in order to improve the sequencing quality in Beckman CEQ2000 automatic DNA sequencer. The most suitable amount of template in DTCS reaction is 125 nanogram; below or above 125 nanogram in template amount will result in low length readable DNA sequence. The sequencing results are better when using 1 micorliter primer than using 0.5 microliter. More length DNA sequences were read when DTCS was carried out in 10 microliter volume than in 5 microliter.