Mitochondrial single nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using cleavable biotinylated dideoxynucleotides.
Mitochondrial single nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using cleavable biotinylated dideoxynucleotides.
复制标题
DOI：
10.1016/j.ab.2012.05.001
复制
发表时间：
2012-08
影响因子：
2.9
通讯作者：
C. Qiu;Shiv Kumar;Jia Guo;Jiesheng Lu;S. Shi;S. Kalachikov;J. Russo;A. Naini;E. Schon;
中科院分区：
文献类型：
--
作者：
C. Qiu;Shiv Kumar;Jia Guo;Jiesheng Lu;S. Shi;S. Kalachikov;J. Russo;A. Naini;E. Schon;
线粒体DNA（mtDNA）单核苷酸多态性（SNP）和突变的表征对于疾病诊断至关重要，这需要准确和灵敏的检测方法和定量，这归因于线粒体异质性。我们在这里报告突变的肌阵挛性癫痫与粗糙红纤维综合征使用化学可裂解的生物素化双脱氧核苷酸和质谱（MS）为基础的固相捕获（SPC）单碱基延伸（SBE）测定的表征。该方法有效地消除了未延伸的引物和引物二聚体，并且碱基和生物素之间可切割的接头的存在允许DNA产物从固相中有效地扩增和释放以用于MS分析。这种方法是能够高复用，和使用不同长度的连接器的每个嘌呤和每个嘧啶允许更好的歧视的四个碱基的MS。这两个同质和异质基因型准确地确定不同的mtDNA样品。通过使用线粒体DNA阴性细胞验证了该方法检测线粒体DNA的特异性。该方法的灵敏度允许检测不到5%的mtDNA异质性水平。这表明基于化学可切割的生物素化双脱氧核苷酸和MS的SPC-SBE方法能够快速、准确和灵敏地进行mtDNA基因分型，并在遗传分析中具有广泛的应用。
Characterization of mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) and mutations is crucial for disease diagnosis, which requires accurate and sensitive detection methods and quantification due to mitochondrial heteroplasmy. We report here the characterization of mutations for myoclonic epilepsy with ragged red fibers syndrome using chemically cleavable biotinylated dideoxynucleotides and a mass spectrometry (MS)-based solid phase capture (SPC) single base extension (SBE) assay. The method effectively eliminates unextended primers and primer dimers, and the presence of cleavable linkers between the base and biotin allows efficient desalting and release of the DNA products from solid phase for MS analysis. This approach is capable of high multiplexing, and the use of different length linkers for each of the purines and each of the pyrimidines permits better discrimination of the four bases by MS. Both homoplasmic and heteroplasmic genotypes were accurately determined on different mtDNA samples. The specificity of the method for mtDNA detection was validated by using mitochondrial DNA-negative cells. The sensitivity of the approach permitted detection of less than 5% mtDNA heteroplasmy levels. This indicates that the SPC–SBE approach based on chemically cleavable biotinylated dideoxynucleotides and MS enables rapid, accurate, and sensitive genotyping of mtDNA and has broad applications for genetic analysis.