Mutation detection, using capillary electrophoresis

Kuypers AWHM, Willems PMW, van der Schans MJ, Linssen PCM, Wessels HMC, de Bruijn CHMM, Everaerts FM, Mensink EJBM (1993) Detection of point mutations in DNA using capillary electrophoresis in a polymer network. J Chromatogr 621 149-156. 

Lou, X. J., Ranaro, N. J., Wilding, R., Fortina, R, and Kricka, L. J., Mutation detection using ligase chain reaction in passivated sdicon-glass microchips and microchip capillary electrophoresis. Biotechniques, 37, 392, 2004. 

The extraordinary complexity of human genes and their products has encouraged the development of extremely high-resolution analytical methods.75 Capillary electrophoresis is competitive with slab gel methods, with resolution up to the order of about 1,000 base pairs for sequencing, sizing, and detection of mutation. Reversed phase HPLC is useful for restriction digest mapping and MALDI-MS up to about 1000 base pairs. 

Tian H, Brody LC, Landers JP. Rapid detection of deletion, insertion, and substitution mutations via heteroduplex analysis using capillary- and microchip-based electrophoresis. Genome Res 2000 10 1403-1413. 

The most widely used genetic screening technique, PCR-RFLP detects a mutation at a specific restriction endonuclease cleavage site at the mutation locus [9], The products from other techniques such as ARMS (amplification refractory mutation system), SSCP (single-strand conformational polymorphism), HPA (heteroduplex polymorphism), and CDCE (constant denaturant capillary electrophoresis) and PCR are usually separable using polymer networks. 

Gao Q, Yeung ES. High-throughput detection of unknown mutations by using multiplexed capillary electrophoresis with poly(vinylpyrrohdone) solution. Anal Chem 2000 72 2499-506. 

Plant DNA analysis has been increasing over the past couple of years because of its ability to pinpoint the origin of drug-related plant material [ 101,102]. Plant STRs are not yet well characterized however, other molecular methods can be combined with capillary gel electrophoresis to establish the identity of plant species [103]. These techniques include amplified fragment length polymorphism (AFLP), random amplification of polymorphic DNA (RAPD), and other similar random primer annealing techniques used for mutation detection. 

Capillary electrophoresis technology has become an indispensable tool for forensic scientists in the biology field since it is able to provide valuable information to aid in the process of law enforcement. The primary application of the technique is in the qualitative analysis of STRs. Isolation of STR mixtures is also possible using relative peak heights. Other applications of CE include quantitative analysis of PCR products, mtDNA sequencing, and mutation detection for the analysis of plant and bacterial DNA. Based on the performance of the methods illustrated above, it is reasonable to expect future researchers and practitioners to continue working to exploit the capabilities of this robust scientific technique and its application to criminal investigations. 

Once the PCR procedure is complete, the products of amplification should be visualized for analysis and interpretation. Agarose gel electrophoresis and ethidium bromide staining can achieve this however, they cannot separate amplification products that differ in size by only a few nucleotides. Polyacrylamide gel electrophoresis or capillary gel electrophoresis can achieve a finer separation (Fig 2.3A,B). PCR amplification followed by gel electrophoresis is frequently used for detection of small deletions or insertions, microsatellite instability, and LOH. For detection of point mutations, the PCR products should be interrogated by other molecular techniques. 

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