Doktycz M J, Arlinghaus H F, Allen R C, Jacobson K B
Biology Division, Oak Ridge National Laboratory, TN 37831-8077.
Electrophoresis. 1992 Aug;13(8):521-8. doi: 10.1002/elps.11501301108.
Alternative protocols are necessary for the use of polyacrylamide gel electrophoresis in genome scale sequencing and mapping studies. The use of radioisotopes and manual gel reading will have to be replaced with a flexible labeling system that can be detected at levels similar or to better than radioisotopes but allows automated, high-speed detection. Labeling with stable isotopes is such an alternative. These nondecaying isotopes have the potential to be detected in sub-attomole quantities, despite being surrounded by the gel matrix, due to the high selectivity and sensitivity of resonance-ionization spectroscopy coupled with a mass spectrometer. In this study the detection limits of sputter-initiated resonance ionization spectroscopy (SIRIS) are investigated using thin, open-faced polyacrylamide gels supported by plastic. This system allows reproducibility and flexibility in the choice of gel size and buffer system since the gel can be cast, washed free of polymerization by-products, dried, and stored until use. Various concentrations of an Sn-labeled oligomer were run on these gels and loads of 5 femtomoles/mm could be detected on a 240 microns thick gel. Gels as thin as 60 microns lower the detectable concentration loads to 1 femtomole/mm. The limiting factor is tin contamination in the gel which, if reduced, will further increase detection. Polymerase chain reaction (PCR) products can also be labeled and detected using Sn isotopes, which could prove useful in mapping studies. Also presented are techniques which will facilitate resolution of these PCR products on open-faced gels by employing discontinuous buffers systems and DNA mobility modifiers.
在基因组规模测序和图谱研究中使用聚丙烯酰胺凝胶电泳需要替代方案。放射性同位素的使用和手动凝胶读数必须被一种灵活的标记系统所取代，该系统能够以与放射性同位素相似或更好的水平进行检测，但允许自动化、高速检测。用稳定同位素进行标记就是这样一种替代方法。这些非衰变同位素尽管被凝胶基质包围，但由于共振电离光谱与质谱仪相结合的高选择性和高灵敏度，有可能在亚阿托摩尔数量级被检测到。在本研究中，使用塑料支撑的薄型、无盖聚丙烯酰胺凝胶研究了溅射引发共振电离光谱（SIRIS）的检测限。该系统在凝胶大小和缓冲系统的选择上具有可重复性和灵活性，因为凝胶可以浇铸、洗涤以去除聚合副产物、干燥并储存直至使用。在这些凝胶上运行了不同浓度的锡标记寡聚物，在240微米厚的凝胶上可以检测到5飞摩尔/毫米的上样量。薄至60微米的凝胶可将可检测的浓度上样量降低至1飞摩尔/毫米。限制因素是凝胶中的锡污染，如果降低该污染，将进一步提高检测能力。聚合酶链反应（PCR）产物也可以用锡同位素进行标记和检测，这在图谱研究中可能会很有用。还介绍了一些技术，这些技术将通过采用不连续缓冲系统和DNA迁移率调节剂来促进这些PCR产物在无盖凝胶上的分离。
