Population-based screening for rare mutations: High-throughput DNA extraction and molecular amplification from Guthrie cards

To determine the population-based frequency of a rare mutation (the 121ins2 mutation in the surfactant protein B gene), we developed high-throughput t echniques to extract reliably and rapidly amplifiable DNA from Guthrie card s. Using a 3-mm punch from each of 10,044 Guthrie cards obtained from, the Missouri Department of Health, we extracted DNA with deionized water by hea ting in the presence of 2% Chelex in a 96-well format. Average yield of DNA from each punch was 52.6 +/- 21 mug. Using 36mer primers and a 10-muL reac tion volume, we amplified a 354-bp fragment of the surfactant protein B gen e that contained the mutation and identified the mutation by its susceptibi lity to restriction enzyme digestion with SfuI The procedure required 5 h p er 96 samples but only 2 h of technician time. The amplification rate on th e first attempt was 99.2%. Based on detection of eight individuals heterozy gous for the mutation (confirmed by direct sequencing), we estimate the all ele frequency to be 0.8/1000 individuals, an estimate not significantly dif ferent from previous estimates based on independent methods. High-throughpu t DNA extraction and amplification will permit establishment of DNA banks a s well as efficient estimation of population-based genotype frequency for b oth rare and common genetic disorders.