Jacobson K B, Arlinghaus H F, Schmitt H W, Sachleben R A, Brown G M, Thonnard N, Sloop F V, Foote R S, Larimer F W, Woychik R P
Biology Research Division, Oak Ridge National Laboratory, Tennessee 37831.
Genomics. 1991 Jan;9(1):51-9. doi: 10.1016/0888-7543(91)90220-9.
The sequencing of DNA by current procedures involves the use of radioisotopic or fluorescent labels. We propose that stable isotopes can be used as such labels and that the large number of stable isotopes available would allow multiplexing so that many DNA segments could be sequenced simultaneously. We have developed methods to use 57Fe2O3 to synthesize ferrocene and to attach the ferrocene to the 5' end of oligonucleotides. The 57Fe-labeled M13 universal primer functioned normally in a Sanger sequencing procedure. When a 57Fe-labeled oligonucleotide had migrated on a polyacrylamide gel it was readily located on the dried gel by scanning with resonance ionization spectroscopy (RIS) coupled with mass spectrometry. Using a 57Fe-labeled primer in a PCR reaction a 2000-bp DNA was produced that was detected by RIS on nylon membrane after agarose electrophoresis. The rapid analysis features of RIS coupled with the multispectral multiplexing possibilities of stable isotopes should significantly increase the rate of determination of DNA sequences.
目前的DNA测序程序需要使用放射性同位素或荧光标记。我们提出稳定同位素可以用作此类标记，并且大量可用的稳定同位素将允许进行多重分析，从而可以同时对许多DNA片段进行测序。我们已经开发出利用57Fe2O3合成二茂铁并将二茂铁连接到寡核苷酸5'端的方法。57Fe标记的M13通用引物在桑格测序程序中正常发挥作用。当57Fe标记的寡核苷酸在聚丙烯酰胺凝胶上迁移后，通过共振电离光谱（RIS）与质谱联用对干燥凝胶进行扫描，很容易找到它。在PCR反应中使用57Fe标记的引物产生了一个2000bp的DNA，在琼脂糖电泳后通过RIS在尼龙膜上检测到。RIS的快速分析特性以及稳定同位素的多光谱多重分析可能性应会显著提高DNA序列的测定速度。
