Null A P, Hannis J C, Muddiman D C
Department of Chemistry, Virginia Commonwealth University, Richmond 23284, USA.
Anal Chem. 2001 Sep 15;73(18):4514-21. doi: 10.1021/ac0103928.
The utility of electrospray ionization Fourier transform ion cyclotron resonance (ESI-FIICR) mass spectrometry as a new approach for genotyping short tandem repeats (STRs) is demonstrated. STRs are currently valued as a powerful source of genetic information with repeats that range in structure from simple to hypervariable. Two tetranucleotide STR loci were chosen to evaluate ESI-FTICR mass spectrometry as a tool for genotyping: HUM-TH01, a simple STR with nonconsensus alleles, and vWA, a compound STR with nonconsensus alleles. For HUM-TH01, the genotype (i.e., repeat number of each allele) was determined for each of 30 individuals using mass measurements of double-stranded amplicons. Low-intensity peaks observed in the spectra of amplicons derived from heterozygous individuals were identified by mass as heteroduplexes that had formed between nonhomologous strands. Mass measurement of the double-stranded vWA amplicon was not sufficient for determining whether the individual was homozygous for allele subtype 18 or 18' since the amplicons differ by only 0.99 Da. Therefore, single-stranded amplicons were generated by incorporating a phosphorylated primer, prepared using T4 polynucleotide kinase, into the PCR phase and subsequently digesting the bottom strand using lambda-exonuclease. Accurate mass measurements were obtained for the single-stranded amplicons using internal calibration and the addition of a correction factor to adjust for the natural variation of isotopic abundances, confirming that the individual is homozygous for allele 18. Our results clearly demonstrate that ESI-FTICR mass spectrometry is a powerful approach to characterize both simple and compound STRs beyond the capabilities of electrophoretic technologies.
本文展示了电喷雾电离傅里叶变换离子回旋共振（ESI-FIICR）质谱作为一种对短串联重复序列（STR）进行基因分型的新方法的实用性。STR目前被视为遗传信息的强大来源，其重复序列的结构从简单到高度可变不等。选择了两个四核苷酸STR位点来评估ESI-FTICR质谱作为基因分型工具：HUM-TH01，一个具有非一致性等位基因的简单STR；以及vWA，一个具有非一致性等位基因的复合STR。对于HUM-TH01，使用双链扩增子的质量测量确定了30个个体中每个个体的基因型（即每个等位基因的重复数）。在杂合个体衍生的扩增子光谱中观察到的低强度峰通过质量鉴定为非同源链之间形成的异源双链体。双链vWA扩增子的质量测量不足以确定个体对于等位基因亚型18或18'是否纯合，因为扩增子仅相差0.99 Da。因此，通过将使用T4多核苷酸激酶制备的磷酸化引物掺入PCR阶段，随后使用λ外切核酸酶消化底部链来产生单链扩增子。使用内部校准和添加校正因子以调整同位素丰度的自然变化，获得了单链扩增子的准确质量测量，证实个体对于等位基因18是纯合的。我们的结果清楚地表明，ESI-FTICR质谱是一种强大的方法，能够表征电泳技术无法实现的简单和复合STR。
