Application of kinetic polymerase chain reaction and molecular beacon assays to pooled analyses and high-throughput genotyping for candidate genes

Min Shi; Diana Caprau; John Dagle; Lene Christiansen; Kaare Christensen; Jeffrey C Murray

doi:10.1002/bdra.10153

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Application of kinetic polymerase chain reaction and molecular beacon assays to pooled analyses and high-throughput genotyping for candidate genes

Journal article

Peer reviewed

Application of kinetic polymerase chain reaction and molecular beacon assays to pooled analyses and high-throughput genotyping for candidate genes

Min Shi, Diana Caprau, John Dagle, Lene Christiansen, Kaare Christensen and Jeffrey C Murray

Birth defects research. A Clinical and molecular teratology, Vol.70(2), pp.65-74

02/2004

DOI: 10.1002/bdra.10153

PMID: 14991913

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Abstract

The addition of DNA analysis to epidemiologic studies that have traditionally incorporated demographic and interview data can provide additional power and open new avenues for investigation. DNA can be obtained from a variety of tissues, but each has attendant variation in sample quantity, quality, and cost of acquisition. Analytic approaches for DNA genotyping are under constant development, but current applications allow small amounts (less than 2 ng per assay) of DNA to be used for genotyping. In this report, we designed effective assays for a spectrum of genes using either kinetic polymerase chain reaction (PCR) or molecular beacon applications. We also investigated the extent to which DNA use and reagent cost could be minimized. Kinetic PCR assays were also applied to investigate the potential of pooled sample analysis. Our results show that small amounts of DNA can be successfully amplified in a high-throughput fashion using both kinetic PCR and molecular beacon methods. Greater than 97% of the genotype results from these two methods are consistent. In addition, error rates in allele frequency measurements using DNA pools of 100 or more samples were often less than 1% and usually less than 3%, which provides another option for substantially minimizing the costs of genotyping in studies involving large numbers of individuals. Effective assays have been designed for a spectrum of genes widely studied in birth defects, including: MTHFR, NAT1, TGFA, RFC1, PAX9, EPHX1, and SKI. An efficient assay has been designed for the detection of the presence of X and Y chromosomes, which can be applied to the studies of sex chromosome abnormalities or sample quality control.

Molecular Probe Techniques

Membrane Proteins - genetics

Humans

Molecular Sequence Data

Genotype

Genetic Testing - methods

Sequence Analysis, DNA

Polymerase Chain Reaction - methods

Carrier Proteins - genetics

Specimen Handling

Methylenetetrahydrofolate Reductase (NADPH2) - genetics

Membrane Transport Proteins

Polymorphism, Single Nucleotide - genetics

Kinetics

Research Design

Details

Title: Subtitle: Application of kinetic polymerase chain reaction and molecular beacon assays to pooled analyses and high-throughput genotyping for candidate genes
Creators: Min Shi - Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242, USA
Diana Caprau
John Dagle
Lene Christiansen
Kaare Christensen
Jeffrey C Murray
Resource Type: Journal article
Publication Details: Birth defects research. A Clinical and molecular teratology, Vol.70(2), pp.65-74
DOI: 10.1002/bdra.10153
PMID: 14991913
NLM abbreviation: Birth Defects Res A Clin Mol Teratol
ISSN: 1542-0752
eISSN: 1542-0760
Publisher: Wiley; United States
Grant note: R01 DE11948 / NIDCR NIH HHS DE08559 / NIDCR NIH HHS U50/CCU 71328 / ODCDC CDC HHS P60 DE13076-02 / NIDCR NIH HHS
Language: English
Date published: 02/2004
Academic Unit: Anatomy and Cell Biology; Stead Family Department of Pediatrics; Epidemiology; Pediatric Dentistry; Craniofacial Anomalies Research Center; Biochemistry and Molecular Biology; Dental Research; Neonatology
Record Identifier: 9984024506802771

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