Hadd A G, Goard M P, Rank D R, Jovanovich S B
Molecular Dynamics/Amersham Pharmacia Biotech, Sunnyvale, CA 94086, USA.
J Chromatogr A. 2000 Oct 13;894(1-2):191-201. doi: 10.1016/s0021-9673(00)00459-3.
DNA sequencing from sub-microliter samples was demonstrated for capillary array electrophoresis by optimizing the analysis of 500 nl reaction aliquots of full-volume reactions and by preparing 500 nl reactions within fused-silica capillaries. Sub-microliter aliquots were removed from the pooled reaction products of 10 microl dye-primer cycle-sequencing reactions and analyzed without modifying either the reagent concentrations or instrument workflow. The impact of precipitation methods, resuspension buffers, and injection times on electrokinetic injection efficiency for 500 nl aliquots were determined by peak heights, signal-to-noise ratios, and changes in base-called readlengths. For 500 nl aliquots diluted to 5 microl in 60% formamide-1 mM EDTA and directly injected, a five-fold increase in signal-to-noise ratios was obtained by increasing injection times from 10 to 80 s without a corresponding increase in peak widths or reduction in readlengths. For 500 nl aliquots precipitated in alcohol, 80 +/- 5% template recovery and a two-fold decrease in conductivity was obtained, resulting in a two-fold increase in peak heights and 50 to 100 bases increase in readlengths. In a comparison of aliquot volumes and precipitation methods, equivalent readlengths were obtained for 500 nl, 4 microl, and 8 microl aliquots by simply adjusting the electrokinetic injection conditions. To ascertain the robustness of this methodology for genomic sequencing, 96 Arabidopsis thaliana subclones were sequenced, with a yield of 38 624 bases obtained from 500 nl aliquots versus 30 764 bases from standard scale reactions. To demonstrate 500 nl sample preparation, reactions were performed in fused-silica capillary reaction chambers using air-based thermal cycling. A readlength of 690 bases was obtained for the polymerase chain reaction product of an Arabidopsis subclone without modifying the reagent concentrations, post-reaction processing or electrokinetic injection workflow. These results demonstrated the fundamental feasibility of small-volume DNA sequencing for high-throughput capillary electrophoresis.
通过优化对全量反应中500 nl反应等分试样的分析，并在熔融石英毛细管内进行500 nl反应，实现了用于毛细管阵列电泳的亚微升样本的DNA测序。从10微升染料引物循环测序反应的合并反应产物中取出亚微升等分试样，在不改变试剂浓度或仪器工作流程的情况下进行分析。通过峰高、信噪比和碱基检出读长的变化，确定了沉淀方法、重悬缓冲液和进样时间对500 nl等分试样电动进样效率的影响。对于在60%甲酰胺-1 mM EDTA中稀释至5微升并直接进样的500 nl等分试样，将进样时间从10秒增加到80秒，信噪比提高了五倍，而峰宽没有相应增加，读长也没有缩短。对于在酒精中沉淀的500 nl等分试样，模板回收率为80±5%，电导率降低了两倍，导致峰高增加了两倍，读长增加了50至100个碱基。在等分试样体积和沉淀方法的比较中，通过简单调整电动进样条件，500 nl、4微升和8微升等分试样获得了相当的读长。为了确定该方法用于基因组测序的稳健性，对96个拟南芥亚克隆进行了测序，从500 nl等分试样中获得了38624个碱基的产量，而标准规模反应获得了30764个碱基。为了展示500 nl样本制备，在熔融石英毛细管反应室中使用基于空气的热循环进行反应。在不改变试剂浓度、反应后处理或电动进样工作流程的情况下，获得了拟南芥亚克隆聚合酶链反应产物690个碱基的读长。这些结果证明了小体积DNA测序用于高通量毛细管电泳的基本可行性。
