Tsuneyoshi T, Ishikawa K, Koga Y, Naito Y, Baba S, Terunuma H, Arakawa R, Prockop D J
Department of Materials Science, Shizuoka Institute of Science and Technology, Japan.
Rapid Commun Mass Spectrom. 1997;11(7):719-22. doi: 10.1002/(SICI)1097-0231(19970422)11:7<719::AID-RCM862>3.0.CO;2-J.
One-base substitution has been detected on the polymerase chain reaction (PCR) products amplified from human mutated DNA for the first time by using mass spectrometry. PCR fragments of 52 base pairs were produced on a collagen gene of an osteogenesis imperfecta patient's heterozygous DNAs. The products were digested with EcoRI restriction enzyme to liberate 3'-end adducts and purified by phenol + chloroform extraction, ammonium acetate addition and ethanol precipitation to remove sodium ions from the phosphoric acid backbone of the DNAs. Purified products were examined using an electrospray ionization mass spectrometer. Mass spectra showed four groups of fragment peaks with the expected molecular masses, which originate from the sense and antisense strands of the heterozygous DNAs.
首次通过质谱法在从人类突变DNA扩增的聚合酶链反应（PCR）产物上检测到单碱基替换。从一名成骨不全患者的杂合DNA的胶原蛋白基因上产生了52个碱基对的PCR片段。产物用EcoRI限制性内切酶消化以释放3'-末端加合物，并通过苯酚+氯仿萃取、添加醋酸铵和乙醇沉淀进行纯化，以从DNA的磷酸主链上去除钠离子。使用电喷雾电离质谱仪检查纯化产物。质谱显示了四组具有预期分子量的片段峰，它们源自杂合DNA的有义链和反义链。
