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

Aaron Hamvas, Michelle Trusgnich, Heather Brice, James Baumgartner, Yuling Hong, Lawrence M. Nogee, F. Sessions Cole

Research output: Contribution to journalArticlepeer-review

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 SfuI. 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.

Original languageEnglish (US)
Pages (from-to)666-668
Number of pages3
JournalPediatric research
Volume50
Issue number5
DOIs
StatePublished - 2001

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health

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