Salas-Solano O, Ruiz-Martinez M C, Carrilho E, Kotler L, Karger B L
Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
Anal Chem. 1998 Apr 15;70(8):1528-35. doi: 10.1021/ac9711448.
In the previous paper, a sample cleanup procedure for DNA sequencing reaction products was developed, in which template DNA was removed by ultrafiltration and the total concentration of salts (chloride and di- and deoxynucleotides) was decreased below 10 microM using gel filtration. In this paper, a quantitative study of the effects of these sample solution components on the injected amount and separation efficiency of the sequencing fragments in capillary electrophoresis is presented. The presence of chloride and deoxynucleotides in a total concentration above 10 microM in the sample solution significantly decreased the amount of DNA sequencing fragments injected into the capillary column. However, the separation efficiency was not affected upon increasing the amount of salt. On the other hand, in the presence of only 0.1 microgram of template in the sample (one-third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentration (approximately 5 microM), the separation efficiency decreased by 70%, and the injected amount of DNA sequencing fragments was 40% lower compared to the sample cleaned by the new purification method. The deleterious effect of template DNA on the separation of sequencing fragments was suppressed in the presence of salt in a concentration above 100 microM in the sample solution. Separately, it was found that both the electric field strength and duration of injection affected the resolution of DNA sequencing fragments when the cleaned up sample solution was used. Separation efficiencies of 15 x 10(6) theoretical plates/m were achieved when the sample was loaded at low electric field, e.g., 25 V/cm for 80 s or less. The results demonstrate that the sample solution components (chloride, deoxynucleotides, template DNA) and injection conditions must be controlled to achieve high performance and rugged DNA sequencing analysis.
在前一篇论文中，开发了一种用于DNA测序反应产物的样品净化程序，其中通过超滤去除模板DNA，并使用凝胶过滤将盐（氯化物以及二脱氧核苷酸和脱氧核苷酸）的总浓度降低至10微摩尔以下。本文对这些样品溶液成分对毛细管电泳中测序片段的进样量和分离效率的影响进行了定量研究。样品溶液中总浓度高于10微摩尔的氯化物和脱氧核苷酸的存在显著降低了注入毛细管柱的DNA测序片段的量。然而，增加盐的量时分离效率不受影响。另一方面，当样品中仅存在0.1微克模板（循环测序中推荐的最低量的三分之一）且氯化物浓度非常低（约5微摩尔）时，分离效率降低了70%，与通过新纯化方法净化的样品相比，DNA测序片段的进样量降低了40%。当样品溶液中盐浓度高于100微摩尔时，模板DNA对测序片段分离的有害影响得到抑制。另外，发现当使用净化后的样品溶液时，电场强度和进样持续时间均会影响DNA测序片段的分辨率。当在低电场下进样，例如在25伏/厘米下进样80秒或更短时间时，分离效率可达15×10⁶理论塔板数/米。结果表明，必须控制样品溶液成分（氯化物、脱氧核苷酸、模板DNA）和进样条件，以实现高性能且稳定的DNA测序分析。
