Capillary Electrophoresis of DNA
Capillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for separation is in the nanogram range. Single‐base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit.
Basic Protocol 1: Separation of Oligonucleotides
Basic Protocol 2: Quantitative PCR Analysis
Figure 10.9.1 Schematic of a CE instrument configured for DNA separations.
Figure 10.9.2 CE separation of a standard poly(A)40‐60 mixture of synthetic oligonucleotides.
Figure 10.9.3 A sizing ladder from a HaeIII digest of φX‐174 DNA. Fragment size in bp is indicated.
Figure 10.9.4 CE separation of a synthetic 20‐mer oligonucleotide. The coupling failure products (shorter migration times) and incomplete deprotection products (longer migration times) are clearly visible.
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|An excellent reference on all aspects of capillary electrophoresis separations. Chapter 11, on DNA, goes into much greater depth than is possible here on the theory of separation, selection of buffers, and selection of gel matrices.|