Abdi Fadi A, Mundt Mark, Doggett Norman, Bradbury E Morton, Chen Xian
Analytical Chemistry Sciences, MS M888, Los Alamos National Laboratory, Los Alamos New Mexico 87545, USA.
Genome Res. 2002 Jul;12(7):1135-41. doi: 10.1101/gr.221402.
We present a mass spectrometry (MS)-based nucleoside-specific mass-tagging method to validate genomic DNA sequences containing ambiguities not resolved by gel electrophoresis. Selected types of (13)C/(15)N-labeled dNTPs are used in PCR amplification of target regions followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-MS analysis. From the mass difference between the PCR products generated with unlabeled nucleosides and products containing (13)C/(15)N-labeled nucleosides, we determined the base composition of the genomic regions of interest. Two approaches were used to verify the target regions: The first approach used nucleosides partially enriched with stable isotopes to identify a single uncalled base in a gel electrophoresis-sequenced region. The second approach used mass tags with 100% heavy nucleosides to examine a GC-rich region of a polycytidine string with an unknown number of cytidines. By use of selected (13)C/(15)N-labeled dNTPs (dCTPs) in PCR amplification of the target region in tandem with MALDI-TOF-MS, we determined precisely that this string contains 11 cytidines. Both approaches show the ability of our MS-based mass-tagging strategy to solve critical questions of sequence identities that might be essential in determining the proper reading frames of the targeted regions.
我们提出了一种基于质谱（MS）的核苷特异性质量标记方法，用于验证包含凝胶电泳无法解析的模糊性的基因组DNA序列。在目标区域的PCR扩增中使用选定类型的（13）C/（15）N标记的dNTP，随后进行基质辅助激光解吸电离飞行时间（MALDI-TOF）-MS分析。根据未标记核苷产生的PCR产物与含有（13）C/（15）N标记核苷的产物之间的质量差异，我们确定了感兴趣的基因组区域的碱基组成。使用两种方法来验证目标区域：第一种方法使用部分富含稳定同位素的核苷来识别凝胶电泳测序区域中单个未确定的碱基。第二种方法使用具有100%重核苷的质量标签来检测具有未知数量胞嘧啶的聚胞嘧啶串的富含GC的区域。通过在目标区域的PCR扩增中串联使用选定的（13）C/（15）N标记的dNTP（dCTP）与MALDI-TOF-MS，我们精确确定该串包含11个胞嘧啶。两种方法都显示了我们基于MS的质量标记策略解决序列同一性关键问题的能力，这些问题在确定目标区域的正确阅读框时可能至关重要。
