Sub-microliter DNA sequencing for capillary array electrophoresis.
Sub-microliter DNA sequencing for capillary array electrophoresis.
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用于毛细管阵列电泳的亚微升 DNA 测序。
DOI：
10.1016/s0021-9673(00)00459-3
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Jovanovich,SB
中科院分区：
文献类型：
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作者：
Hadd,AG;Goard,MP;Rank,DR;Jovanovich,SB
亚微升样品的DNA测序通过优化500nl满体积反应等分的分析和在熔融二氧化硅毛细管内制备500nl反应，证明了毛细管阵列电泳。从10 μl染料-引物循环-测序反应的混合反应产物中去除亚微升等分，在不改变试剂浓度或仪器工作流程的情况下进行分析。沉淀方法、再悬浮缓冲液和注射次数对500 nl等分液电动注射效率的影响通过峰高、信噪比和碱基（称为读长）的变化来确定。在60%的甲酰胺- 1 mM EDTA溶液中，将500 nl溶液稀释至5 μl后直接注射，注射时间从10 s增加到80 s，信噪比增加了5倍，而峰宽没有相应增加，读长也没有相应减少。在酒精中沉淀500nl时，模板回收率为80±5%，电导率降低2倍，导致峰高增加2倍，读长增加50 ~ 100个碱基。在等分体积法和沉淀法的比较中，通过简单调整电动进样条件，在500 μl、4 μl和8 μl等分体积下获得了等效的读长。为了确定该方法在基因组测序方面的稳健性，对96个拟南芥亚克隆进行了测序，从500个等分中获得38624个碱基，而标准规模反应获得30764个碱基。为了演示500nl样品的制备，在熔融二氧化硅毛细管反应室中使用空气基热循环进行反应。在不改变试剂浓度、反应后处理和电动注射流程的情况下，获得了一个拟南芥亚克隆聚合酶链反应产物的全长690个碱基。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。
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 μl 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 μl 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 μl, and 8 μl 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.