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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242, USA.
Min
M
Shi
Diana
D
Caprau
John
J
Dagle
Lene
L
Christiansen
Kaare
K
Christensen
Jeffrey C
JC
Murray
D002352:Carrier Proteins / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012107:Research Design; D017422:Sequence Analysis, DNA; D013048:Specimen Handling
D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C120043:SLC19A2 protein, human; D042965:Methylenetetrahydrofolate Reductase (NADPH2)