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 Nogee, F. Sessions Cole

Research output: Contribution to journalArticle

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
StatePublished - 2001

Fingerprint

Mutation
DNA
Population
Surface-Active Agents
Inborn Genetic Diseases
Gene Frequency
Heating
Genes
Digestion
Genotype
Water
Health
Enzymes
IgA receptor

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health

Cite this

Hamvas, A., Trusgnich, M., Brice, H., Baumgartner, J., Hong, Y., Nogee, L., & Sessions Cole, F. (2001). Population-based screening for rare mutations: High-throughput DNA extraction and molecular amplification from Guthrie cards. Pediatric Research, 50(5), 666-668.

Population-based screening for rare mutations : High-throughput DNA extraction and molecular amplification from Guthrie cards. / Hamvas, Aaron; Trusgnich, Michelle; Brice, Heather; Baumgartner, James; Hong, Yuling; Nogee, Lawrence; Sessions Cole, F.

In: Pediatric Research, Vol. 50, No. 5, 2001, p. 666-668.

Research output: Contribution to journalArticle

Hamvas, A, Trusgnich, M, Brice, H, Baumgartner, J, Hong, Y, Nogee, L & Sessions Cole, F 2001, 'Population-based screening for rare mutations: High-throughput DNA extraction and molecular amplification from Guthrie cards', Pediatric Research, vol. 50, no. 5, pp. 666-668.
Hamvas, Aaron ; Trusgnich, Michelle ; Brice, Heather ; Baumgartner, James ; Hong, Yuling ; Nogee, Lawrence ; Sessions Cole, F. / Population-based screening for rare mutations : High-throughput DNA extraction and molecular amplification from Guthrie cards. In: Pediatric Research. 2001 ; Vol. 50, No. 5. pp. 666-668.
@article{b4be6dfed4d9467aae23acb869667311,
title = "Population-based screening for rare mutations: High-throughput DNA extraction and molecular amplification from Guthrie cards",
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.",
author = "Aaron Hamvas and Michelle Trusgnich and Heather Brice and James Baumgartner and Yuling Hong and Lawrence Nogee and {Sessions Cole}, F.",
year = "2001",
language = "English (US)",
volume = "50",
pages = "666--668",
journal = "Pediatric Research",
issn = "0031-3998",
publisher = "Lippincott Williams and Wilkins",
number = "5",

}

TY - JOUR

T1 - Population-based screening for rare mutations

T2 - High-throughput DNA extraction and molecular amplification from Guthrie cards

AU - Hamvas, Aaron

AU - Trusgnich, Michelle

AU - Brice, Heather

AU - Baumgartner, James

AU - Hong, Yuling

AU - Nogee, Lawrence

AU - Sessions Cole, F.

PY - 2001

Y1 - 2001

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0034770395&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034770395&partnerID=8YFLogxK

M3 - Article

C2 - 11641464

AN - SCOPUS:0034770395

VL - 50

SP - 666

EP - 668

JO - Pediatric Research

JF - Pediatric Research

SN - 0031-3998

IS - 5

ER -