High-throughput screening for the detection of unknown mutations: improved productivity using heteroduplex analysis.

The detection of specific diseasecausing mutations in DNA is an important adjunct to successful genetic counseling in families with a history of genetic disorders and for prenatal diagnosis. Researchers performing routine mutation detection on large multi-exonic disease genes face a huge and laborious task, especially in the absence of regional clustering of mutations within the gene. Neurofibromatosis Type 1 (NF1) affects more than 20 000 people in Britain alone, however, the causative mutations in the vast majority are unknown. The NF1 gene comprises 60 exons and covers a genomic region of 350 kb (10). Previous studies indicate that screening all 60 exons might be necessary to ascertain more about the type of mutations present and the possible genetic mechanisms involved in causing the condition. The labor-intensive nature of many current molecular biological techniques is all too apparent, and although dramatic advances in automation have recently been achieved, their cost makes them inaccessible to many researchers. Since a large number of samples must be analyzed quickly and economically, an accurate, high-throughput mutation detection method is essential. A well-designed robust assay should provide the basis for a large screening program, and some consideration was given to the various options available. Reviews (4,5) indicate that unfortunately, many of the most sensitive mutation detection techniques (95%–99% detection rate) are also the most laborintensive, with low sample throughput, and are usually complex and costly. In addition, they often require purification, restriction enzyme digestion, involve the use of radiochemicals or toxic chemicals and utilize expensive specialized equipment. Single-stranded conformation polymorphism (SSCP) (12) and heteroduplex analysis (HA), first described by Nagamine et al. (11), although not being the most sensitive methods (75%–90% mutation detection), have few of the above disadvantages. The major limitation of these two techniques, however, is the small size range of the polymerase chain reaction (PCR) products that give maximum sensitivity; 100–300 bp for SSCP (6) and 100–400 bp for HA (2), with loss of sensitivity outside these ranges. Thus, HA would appear to be the most suitable method for large-scale screening for mutations in the NF1 gene in which the size range of the exons give PCR products from 190–549 bp. This method has been used to detect 9 novel mutations in a 549-bp fragment (10) and many others identified in smaller PCR fragments (1,16,17). We investigated whether sample throughput could be greatly increased without adversely affecting the sensitivity of the HA technique. MDE gel (AT Biochem, Malvern, PA, USA) is a unique polyacrylamidebased matrix that has been shown to give improved resolution and reproducibility in the detection of both SSCP and heteroduplex bands (3,15). Gels are subsequently visualized by silver staining, which is a more sensitive method than ethidium bromide staining (9). The most time-consuming component of this method is the silver-staining step, thus if the number of samples analyzed per gel could be dramatically increased, fewer gels would be required, therefore greatly increasing cost effectiveness. Previous attempts to improve sample throughput have involved the pooling of DNA samples (8) or the performance of multiplex PCRs (13). Optimal resolution on MDE gels is obtained by electrophoresing DNA at least 30 cm through a 40-cm-long gel. Two PCR products were combined in each pool; any more may compromise sensitivity. Heteroduplex bands can be missed when analyzed on a 0.4-mmthick gel because of their reduced intensity compared to the homoduplex band (2); therefore, samples are run on a thicker gel. Using a 32-tooth, wellforming comb, pooled samples were run in 30 wells (60 separate PCRs), with the two outer wells reserved, one for a 1-kb DNA ladder (Life Technologies, Paisley, Scotland, UK) and the other for a positive control sample (known mutation). Instead of performing complex multiplex PCRs, which is possible for HA, we investigated the use of sequential loadings. Heteroduplexes usually migrate slower than the corresponding homoduplexes, forming mutant bands less than 1 cm away, and