MANIFOLD SEQUENCING - EFFICIENT PROCESSING OF LARGE SETS OF SEQUENCING REACTIONS
MANIFOLD SEQUENCING - EFFICIENT PROCESSING OF LARGE SETS OF SEQUENCING REACTIONS
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DOI：
10.1073/pnas.91.6.2245
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发表时间：
1994-03-15
影响因子：
11.1
通讯作者：
LANDEGREN, U
中科院分区：
文献类型：
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作者：
LAGERKVIST, A;STEWART, J;LANDEGREN, U
用于DNA测序的自动化仪器大大简化了桑格测序程序中的数据收集。相比之下，制备测序模板、进行测序反应和将这些加载到仪器上的所谓前端问题仍然是大规模测序项目的主要障碍。我们在此描述了使用歧管支持物来制备并在大量模板上并行进行测序反应，以及将反应产物加载到测序仪器上。以这种方式，所有反应步骤都在不移液样品的情况下进行。该策略适用于测序PCR扩增克隆的人线粒体D-环和检测杂合位置的人主要组织相容性复合体II类基因HLA-DQB，从基因组DNA样本扩增。该技术将促进临床背景下的测序，并可能形成更有效的基因组测序策略的基础。
Automated instruments for DNA sequencing greatly simplify data collection in the Sanger sequencing procedure. By contrast, the so-called front-end problems of preparing sequencing templates, performing sequencing reactions, and loading these on the instruments remain major obstacles to extensive sequencing projects. We describe here the use of a manifold support to prepare and perform sequencing reactions on large sets of templates in parallel, as well as to load the reaction products on a sequencing instrument. In this manner, all reaction steps are performed without pipetting the samples. The strategy is applied to sequencing PCR-amplified clones of the human mitochondrial D-loop and for detection of heterozygous positions in the human major histocompatibility complex class II gene HLA-DQB, amplified from genomic DNA samples. This technique will promote sequencing in a clinical context and could form the basis of more efficient genomic sequencing strategies.