Mengel-Jørgensen J, Sanchez J J, Børsting C, Kirpekar F, Morling N
Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, 11 Frederik V's Vej, DK-2100 Copenhagen Ø, Denmark.
Anal Chem. 2005 Aug 15;77(16):5229-35. doi: 10.1021/ac0502044.
A novel single-base extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted of only deoxyribonucleotides (hereafter denoted standard primers). Biotin-labeled ddNTPs were used in the SBE reaction, and the SBE products were purified using the monomeric avidin triethylamine purification protocol, ensuring that only primers extended with a biotin-ddNTP in the 3'-end were isolated. A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The cleaved SBE products were detected in the 2000-5500 m/z range, and the noncleaved SBE products were detected in the 5500-10 000 m/z range. The method was validated by typing 17 Y chromosome single-nucleotide polymorphisms in 100 males with a 17-plex SBE package containing 9 chimeric primers and 8 standard primers.
本文描述了一种新型单碱基延伸（SBE）分析方法，该方法在同一反应混合物中使用可切割和不可切割的SBE引物。可切割的SBE引物由脱氧核糖核苷酸和一个核糖核苷酸组成（以下简称嵌合引物），而不可切割的SBE引物仅由脱氧核糖核苷酸组成（以下简称标准引物）。SBE反应中使用生物素标记的双脱氧核苷酸三磷酸（ddNTPs），并使用单体抗生物素蛋白三乙胺纯化方案纯化SBE产物，以确保仅分离出3'端用生物素-ddNTP延伸的引物。开发了一种核糖核酸酶混合物，可特异性切割嵌合引物，而不考虑核糖核苷酸的碱基，而不含核糖核苷酸的标准引物不受核糖核酸酶处理的影响。使用基质辅助激光解吸/电离飞行时间质谱仪以线性模式分析SBE产物。在2000-5500 m/z范围内检测到切割后的SBE产物，在5500-10000 m/z范围内检测到未切割的SBE产物。通过使用包含9个嵌合引物和8个标准引物的17重SBE试剂盒对100名男性的17个Y染色体单核苷酸多态性进行分型，验证了该方法。
