Oberacher H, Oefner P J, Hölzl G, Premstaller A, Davis K, Huber C G
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innrain 52a, A-6020 Innsbruck, Austria.
Nucleic Acids Res. 2002 Jul 15;30(14):e67. doi: 10.1093/nar/gnf066.
Allelic discrimination of single nucleotide polymorphisms (SNPs) and, particularly, determination of the phase of multiple variations are of utmost importance in genetics. The physicochemical separation of alleles by completely denaturing ion-pair reversed-phase high-performance liquid chromatography and their on-line sequence determination by electrospray ionization mass spectrometry is demonstrated. Simultaneous genotyping of two and three simple sequence polymorphisms contained within 73-114 bp was accomplished with low femtomolar amounts of unpurified amplicons from polymerase chain reaction. Determination of allelic composition is enabled by the high accuracy (better than 0.019%) of intact mass measurements or by comparative sequencing using gas-phase fragmentation and tandem mass spectrometry in combination with fully automated, computer-aided data interpretation.
单核苷酸多态性（SNP）的等位基因鉴别，尤其是多个变异相位的确定在遗传学中至关重要。本文展示了通过完全变性离子对反相高效液相色谱法对等位基因进行物理化学分离，并通过电喷雾电离质谱法进行在线序列测定。利用聚合酶链反应产生的低飞摩尔量未纯化扩增子，实现了对73 - 114 bp内包含的两个和三个简单序列多态性的同时基因分型。完整质量测量的高精度（优于0.019%）或使用气相碎裂和串联质谱法结合全自动计算机辅助数据解释的比较测序，能够实现等位基因组成的测定。
