Abstract
To determine the population-based frequency of a rare mutation (the 121ins2 mutation in the surfactant protein B gene), we developed high-throughput techniques to extract reliably and rapidly amplifiable DNA from Guthrie cards. 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 heating in the presence of 2% Chelex in a 96-well format. Average yield of DNA from each punch was 52.6 ± 21 μg. Using 36mer primers and a 10-μL reaction volume, we amplified a 354-bp fragment of the surfactant protein B gene that contained the mutation and identified the mutation by its susceptibility to restriction enzyme digestion with Sfu I. The procedure required 5 h per 96 samples but only 2 h of technician time. The amplification rate on the first attempt was 99.2%. Based on detection of eight individuals heterozygous for the mutation (confirmed by direct sequencing), we estimate the allele frequency to be 0.8/1000 individuals, an estimate not significantly different from previous estimates based on independent methods. High-throughput DNA extraction and amplification will permit establishment of DNA banks as well as efficient estimation of population-based genotype frequency for both rare and common genetic disorders.
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Abbreviations
- SP-B:
-
surfactant protein B
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Acknowledgements
The authors thank Corrine Hamvas, Jennifer Heeley, Robert Jones, Qua Mei Lei, Robin Peterson, George Roffman, Thomas Sandbothe, and Jasmine Wong for technical assistance.
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Supported in part by a grant from the National Institutes of Health (HL/HD 54187; F.S.C.) and the Eudowood Foundation (L.M.N.).
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Hamvas, A., Trusgnich, M., Brice, H. et al. Population-Based Screening for Rare Mutations: High-Throughput DNA Extraction and Molecular Amplification from Guthrie Cards. Pediatr Res 50, 666–668 (2001). https://doi.org/10.1203/00006450-200111000-00021
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DOI: https://doi.org/10.1203/00006450-200111000-00021
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