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  • Review Article
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DNA Pooling: a tool for large-scale association studies

Key Points

  • DNA pooling is an effective way of reducing the cost of genotyping in large-scale association studies.

  • DNA pools should be constituted with care to ensure that equal amounts of DNA are contributed by the individuals that make up a pool.

  • Accurate quantitative genotyping assays are available for use on pooled DNA.

  • Differential amplification occurs for many single-nucleotide polymorphisms, and this bias should be corrected in the estimation of allele frequency from pooled DNA.

  • A two-stage design, in which positive marker loci from pooling studies are followed by confirmatory individual genotyping, might represent the best trade-off between the cost savings of pooling and the full information that is provided by individual genotyping.

  • Random experimental errors in the constitution of DNA pools and in the measurement of allele frequencies from pooled DNA should be taken into account in statistical analysis.

  • Sophisticated pooling designs are being developed that can take account of hidden population stratification, confounders and interactions, and that allow the analysis of haplotypes.

Abstract

DNA pooling is a practical way to reduce the cost of large-scale association studies to identify susceptibility loci for common diseases. Pooling allows allele frequencies in groups of individuals to be measured using far fewer PCR reactions and genotyping assays than are used when genotyping individuals. Here, we discuss recent developments in quantitative genotyping assays and in the design and analysis of pooling studies. Sophisticated pooling designs are being developed that can take account of hidden population stratification, confounders and inter-loci interactions, and that allow the analysis of haplotypes.

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Figure 1: A protocol for constructing DNA pools.
Figure 2: A two-stage pooling study to identify quantitative trait loci.

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Acknowledgements

P.S. was supported by grants from the UK Medical Research Council, the Wellcome Trust and the National Eye Institute.

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Correspondence to Pak Sham.

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DATABASES

LocusLink

HLA class II DR

HLA class II DQ

OMIM

type I (insulin-dependent) diabetes mellitus

FURTHER INFORMATION

Joel Bader's lab

Pak Sham's lab

Pyrosequencing

Glossary

STUTTER BANDS

The signals that indicate the presence of DNA fragments that are one or two repeats shorter than the true allele, owing to a 'slippage' artefact that arises from the PCR reaction.

FLUORIMETRY

An assay for measuring DNA concentration in which a fluorescent dye is used that intercalates quantitatively between stacked DNA base pairs.

QUANTITATIVE REAL-TIME PCR

A procedure in which the PCR reaction is tracked as it progresses, by monitoring the accumulating signal that is provided by a fluorescent dye released during each PCR cycle.

MALDI–TOF

A mass spectrometry method in which laser-vaporized PCR fragments are accelerated through a vacuum using an electric field, eventually having an impact on a detector. The time taken for the fragments to travel the distance from the plate to the detector is measured and depends on the charge-to-mass ratio of each molecule, so providing a way to distinguish between allele-specific products.

TAQMAN™

A proprietary system that allows the progression of a PCR reaction to be monitored in real time.

QUANTITATIVE TRAIT

A measurable trait that depends on the cumulative action of many genes and that can vary among individuals over a given range to produce a continuous distribution of phenotypes. Common examples include height, weight and blood pressure.

PEARSON X2-TEST

A statistical test that is used to assess whether the frequencies of individuals in different categories of one or more qualitative variables are consistent with those frequencies that are predicted under a certain hypothesis.

QUALITATIVE TRAIT

Those traits for which there is a sharp distinction between phenotypes — the trait is usually present or not. Often only one or a few genes are involved in the expression of qualitative traits.

RELATIVE RISK

The ratio of the risk of developing a disease in individuals who have been exposed to a risk factor to that in individuals who have not been exposed to the risk factor.

POPULATION STRATIFICATION

The presence of multiple population subgroups that show limited inter-breeding. When such subgroups differ both in allele frequency and in disease prevalence, this can lead to erroneous results in association studies.

HAPLOTYPE

The allelic configuration of two or more alleles on a single chromosome of a given individual.

LINKAGE DISEQUILIBRIUM

This occurs when the frequency of a particular haplotype for two or more loci deviates significantly from that expected from the product of the observed allelic frequencies at each locus.

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Sham, P., Bader, J., Craig, I. et al. DNA Pooling: a tool for large-scale association studies. Nat Rev Genet 3, 862–871 (2002). https://doi.org/10.1038/nrg930

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