Soper S A, Williams D C, Xu Y, Lassiter S J, Zhang Y, Ford S M, Bruch R C
Department of Chemistry, Louisiana State University, Baton Rouge 70803-1804, USA.
Anal Chem. 1998 Oct 1;70(19):4036-43. doi: 10.1021/ac980288z.
A miniaturized, solid-phase nanoreactor was developed to prepare Sanger DNA-sequencing ladders which was directly interfaced to a capillary gel electrophoresis system. A biotinylated fragment of the rat brain actin gene (1 kbp) was amplified by PCR and attached to the interior wall of an (aminoalkyl)silane-derivatized fused-silica capillary tube via a biotin/streptavidin/biotin linkage. Coverage of the capillary wall with the biotinylated DNA averaged 77 +/- 10%. Stability of the anchored template under pressure (33 nL/s) and electroosmotic flows (11.3 nL/s) were favorable, requiring rinsing for > 150 h to reduce the surface coverage by only 50%. In addition, the immobilized template was stable toward temperatures required for preparing sequencing ladders, even under cycling conditions. Standard Sanger dideoxynucleotide termination performed in a large-volume (approximately 8 microL) solid-phase reactor using the thermally stable polymerase enzymes Taq and Vent and the polymerases T7 and Bst with off-line slab gel electrophoresis and autoradiographic detection indicated that acceptable fragment generation was achieved only in the case of the thermally stable polymerases. Banding was not apparent for T7 and Bst since all reagents were inserted into the column in a single plug at the beginning of the reaction. A small volume reactor (volume approximately 62 nL) was then used to perform DNA polymerase reactions and was coupled directly to a capillary gel column for separation. The capillary reactor was placed inside a thermocycler to control the temperature during chain extension and was directly connected to the gel column via zero dead volume fused-silica connectors. The complementary DNA fragments generated (C-track only) in the reactor were denatured using heat and directly injected onto the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. Extension and single-base separation resolution of the C-track, which was directly injected onto the gel column, was estimated to be > 450 bases from the primer annealing site with plate numbers ranging from 1 x 10(6) to 2 x 10(6)/m.
一种小型化的固相纳米反应器被开发用于制备桑格DNA测序梯，该反应器直接与毛细管凝胶电泳系统相连。大鼠脑肌动蛋白基因的生物素化片段（1千碱基对）通过聚合酶链式反应（PCR）扩增，并通过生物素/链霉亲和素/生物素连接附着在（氨基烷基）硅烷衍生化的熔融石英毛细管内壁上。生物素化DNA在毛细管内壁的覆盖率平均为77±10%。锚定模板在压力（33纳升/秒）和电渗流（11.3纳升/秒）下的稳定性良好，冲洗超过150小时后表面覆盖率才仅降低50%。此外，即使在循环条件下，固定化模板对于制备测序梯所需的温度也很稳定。在使用热稳定聚合酶Taq和Vent以及聚合酶T7和Bst的大容量（约8微升）固相反应器中进行标准桑格双脱氧核苷酸终止反应，并采用离线平板凝胶电泳和放射自显影检测，结果表明只有在热稳定聚合酶的情况下才能获得可接受的片段生成。对于T7和Bst，由于所有试剂在反应开始时以单个塞子的形式插入柱中，条带不明显。然后使用小体积反应器（体积约62纳升）进行DNA聚合酶反应，并直接与毛细管凝胶柱相连进行分离。毛细管反应器置于热循环仪内以控制链延伸过程中的温度，并通过零死体积的熔融石英连接器直接连接到凝胶柱。反应器中生成的互补DNA片段（仅C泳道）通过加热变性，然后直接注入填充凝胶的毛细管进行大小分离，使用近红外激光诱导荧光进行检测。直接注入凝胶柱的C泳道的延伸和单碱基分离分辨率估计从引物退火位点起大于450个碱基，塔板数范围为1×10⁶至2×10⁶/米。
