Ross P L, Belgrader P
Advanced DNA Technology Development Branch, Armed Forces Institute of Pathology, Rockville, Maryland 20850, USA.
Anal Chem. 1997 Oct 1;69(19):3966-72. doi: 10.1021/ac970312t.
The analysis of an important class of human genetic polymorphisms, short tandem repeats (STRs), using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described. Several model STR systems have been investigated to evaluate MALDI-TOFMS as a realistic alternative to established electrophoresis procedures, and to develop rapid and generally applicable approaches to polymerase chain reaction (PCR) product purification for MALDI-TOFMS analysis. A purification/preconcentration method for PCR product preparation based on affinity capture of biotin-labeled PCR products is demonstrated to be directly compatible with MALDI-TOFMS analysis. The entire sample preparation for MALDI-TOFMS analysis immediately following PCR amplification from human DNA extracts can be accomplished routinely in under 12 min in a single Eppendorf tube. The simplicity of this approach essentially eliminates the sample preparation bottleneck encountered with MALDI-TOFMS analysis of PCR products using existing methods. Using this method, encouraging genotyping results are demonstrated for the THO1 and TPOxx STR systems using subpicomole quantities that represent a fraction of the original dsDNA from a single PCR reaction. The technique is also demonstrated to facilitate rapid sizing of PCR fragments larger than 200 bases using MALDI-TOFMS. As described here, the analysis of DNA can be accomplished in a manner that takes advantage of the rapid and accurate analysis capabilities offered by MALDI-TOFMS.
本文描述了使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）对一类重要的人类遗传多态性——短串联重复序列（STR）进行分析。已对多个STR模型系统进行了研究，以评估MALDI-TOFMS作为现有电泳方法的切实可行替代方法，并开发用于MALDI-TOFMS分析的快速且普遍适用的聚合酶链反应（PCR）产物纯化方法。一种基于生物素标记的PCR产物亲和捕获的PCR产物制备纯化/预浓缩方法被证明与MALDI-TOFMS分析直接兼容。从人DNA提取物进行PCR扩增后紧接着进行MALDI-TOFMS分析的整个样品制备过程，通常可在单个Eppendorf管中于12分钟内完成。这种方法的简便性基本上消除了使用现有方法对PCR产物进行MALDI-TOFMS分析时遇到的样品制备瓶颈。使用该方法，对于THO1和TPOxx STR系统，使用亚皮摩尔量（代表单个PCR反应中原始双链DNA的一小部分）展示了令人鼓舞的基因分型结果。该技术还被证明有助于使用MALDI-TOFMS对大于200个碱基的PCR片段进行快速大小测定。如此处所述，DNA分析可以利用MALDI-TOFMS提供的快速且准确的分析能力来完成。
