The accurate detection of sequence variants is an essential tool in both diagnostic and research-based molecular genetics. Currently, the most widely used technique is Single Stranded Conformational Polymorphism (SSCP) analysis. Sensitivity of SSCP ranges from 70-95 % but drops by 50 % when the PCR product size increases above 400 base pairs (bp). There have been many reports of improved sensitivity by altering several parameters and this technique relies heavily on experimental optimisation of conditions to improve resolution.
The Multiphor II electrophoresis system (GE Healthcare, formerly Amersham Biosciences) is a two buffer-based, temperature controlled, horizontal gel system. The system was previously reported as functioning optimally using a single defined SSCP condition, with a detection rate of 97.5 % and the capability of extending mutation analysis to 500-600 bp fragments. It also allows heteroduplex (HD) analysis to be used in conjunction with SSCP analysis (SSCP-HD analysis). This system and its single defined PCR and SSCP-HD condition was tested in this laboratory.
The ATP binding cassette transporter (ABCA4) gene was screened for sequence variants and 57 potential disease-associated alleles were identified. Only 2 different conditions were required to resolve these variants on the Multiphor II system; 9 oC or 12 oC. The gel-running temperatures were determined empirically for each fragment. On the other hand, detection of 16 known sequence variations of the Rhodopsin (RHO) gene required much optimisation and deviation from the previously published condition. Samples were resolved by altering parameters such as PCR cycling conditions, electrophoresis time, electrophoresis temperature, and the ratio of PCR product to loading dye. One amplicon (284 bp) in particular required three SSCP-HD conditions in order to achieve adequate resolution. This emphasises the fact that the sequence flanking a variation is a major contributor to the sensitivity of SSCP analysis.
In addition, we report a dual application of this particular gel system - rapid resolution of small DNA fragments, generated by restriction endonuclease digestion. This system enabled the separation of 128 bp, 112 bp and 16 bp fragments of the brain-derived neurotrophic factor (BDNF) gene after XhoI digestion. In addition, 169 bp, 143 bp and 26 bp fragments of the catechol-O-methyltransferase (COMT) gene were resolved after Hsp92II digestion.
In conclusion, we have found that although the conditions for sequence variation detection using the Multiphor II system require more optimisation than previously reported, the system is very efficient, cost effective and sensitive and can serve the dual purpose of separating DNA fragments based on their size, more efficiently than agarose gel electrophoresis. Visualisation is non-radioactive, the gels are small and easy to handle, and temperature can be controlled and manipulated to improve sensitivity and reproducibility.
L. Roberts, A.V. September, C. L. Cupido, O. Matthysen, R. Ramesar, J. Greenberg
MRC/UCT Human Genetics Research Unit
Division of Human Genetics
Department of Clinical Laboratory Sciences
University of Cape Town