Walters J J, Muhammad W, Fox K F, Fox A, Xie D, Creek K E, Pirisi L
Department of Microbiology and Immunology, University of South Carolina, School of Medicine, Columbia, SC 29208, USA.
Rapid Commun Mass Spectrom. 2001;15(18):1752-9. doi: 10.1002/rcm.435.
Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.
在编码肿瘤抑制蛋白p53的基因中，单核苷酸多态性（SNP）和突变都很常见。SNP发生在基因内的特定位置，而突变可能分布在基因的大片段区域。在确定核苷酸差异时，除了桑格测序法外，质谱分析法是唯一能提供直接结构信息的方法。在进行简单纯化和在线加热以限制离子加合后，对完整的聚合酶链反应（PCR）产物进行电喷雾电离（ESI）四极杆质谱（MS）分析。与我们之前的工作不同，PCR产物是直接从基因组DNA而非质粒中扩增得到的。对p53基因的两种已知多态性进行了基因分型。在ESI四极杆质谱分析中，通过40.0 Da的变化分别检测到了胞嘧啶（C）或鸟嘌呤（G）的颠换，即C <--> G（互补链上为G <--> C）。以已知的PCR产物作为标准，对10个人类样本确定的基因型与限制性片段长度多态性（RFLP）分析结果相符。胞嘧啶/胸腺嘧啶（T）的转换，即C <--> T（互补链上为G <--> A），也通过ESI-MS进行了基因分型。这种SNP在一条链上（C <--> T）通过15.0 Da的变化来区分，在另一条链上（G <--> A）通过16.0 Da的变化来区分。适当的样品制备和仪器配置（包括加热的进样针和质谱源）对于成功进行完整PCR产物的ESI四极杆质谱分析至关重要，以限制加合物的形成。
