Classic mutation detection methods

The hemoglobinopathies were the first genetic diseases to be characterized at the molecular level and have been used as a prototype for the development of almost every new technique of mutation detection for more than 20 years. Consequently there are numerous PCR-based techniques described in the literature that can be used for PND of the globin gene mutations. The most commonly used techniques include: allele specific oligonucleotide (ASO) hybridization or dot blot analysis, reverse dot blot analysis, the amplification refractory mutation system (ARMS), denaturing gradient gel electrophoresis (DGGE), gap PCR, restriction fragment length polymorphism (RFLP) analysis and direct DNA sequencing (Old et al., 2000; Kanavakis et al., 1997). Each technique has its relative advantages and disadvantages in terms of simplicity, speed, and cost. The choice of techniques used by any laboratory will be influenced by the infrastructure and expertise available in the laboratory, the budget, the sample throughput, and the spectrum of mutations in the target population. In any laboratory providing PND, it is recommended that at least two alternative methods are available for validating each mutation in every prenatal sample.

Without doubt, the provision of PND requires the highest standards in laboratory practice to minimize sources of potential errors and ensure an accurate result. Based on more than two decades of experience, a general consensus has been reached on the precautionary measures to minimize risk of diagnostic errors (Old et al., 2000; Traeger-Synodinos et al., 2002).

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