Limits...
Analysis of copy number variation using quantitative interspecies competitive PCR.

Williams NM, Williams H, Majounie E, Norton N, Glaser B, Morris HR, Owen MJ, O'Donovan MC - Nucleic Acids Res. (2008)

Bottom Line: Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility.Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts.In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychological Medicine, Wales School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. williamsnm@cf.ac.uk

ABSTRACT
Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

Show MeSH

Related in: MedlinePlus

Schematic representation of the principle of quantitative interspecies competitive PCR. Human and P. troglodytes specific alleles are represented by alleles ‘C’ and ‘G’, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553599&req=5

Figure 1: Schematic representation of the principle of quantitative interspecies competitive PCR. Human and P. troglodytes specific alleles are represented by alleles ‘C’ and ‘G’, respectively.

Mentions: We have applied competitive PCR to determine DNA copy number by exploiting the high degree of conservation between orthologous human and chimpanzee genomic sequence. To achieve this we have co-amplified orthologous loci and targeted non-conserved nucleotides by primer extension to distinguish between template from the two species (Figure 1). Quantitative interspecies qicPCR uses the entire genome of a single chimpanzee as a competitor. This results in the requirement for only a single competitor sample for all assays and dramatically increasing the potential of large numbers of loci to be analysed in multiplex. As the rate of fixed (non-polymorphic) interspecies sequence divergence is ∼1% (26) then >3 × 107 single nucleotides could potentially be targeted. Therefore, qicPCR can potentially be used to analyse the majority of CNVs. These factors, together with accuracy, low cost and potential application to array platforms make it potentially amenable to studies performing multiplex high throughput association analysis of CNVs. Here, we establish proof of concept by first accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of qicPCR to analyse CNVs in association studies by genotyping chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.Figure 1.


Analysis of copy number variation using quantitative interspecies competitive PCR.

Williams NM, Williams H, Majounie E, Norton N, Glaser B, Morris HR, Owen MJ, O'Donovan MC - Nucleic Acids Res. (2008)

Schematic representation of the principle of quantitative interspecies competitive PCR. Human and P. troglodytes specific alleles are represented by alleles ‘C’ and ‘G’, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553599&req=5

Figure 1: Schematic representation of the principle of quantitative interspecies competitive PCR. Human and P. troglodytes specific alleles are represented by alleles ‘C’ and ‘G’, respectively.
Mentions: We have applied competitive PCR to determine DNA copy number by exploiting the high degree of conservation between orthologous human and chimpanzee genomic sequence. To achieve this we have co-amplified orthologous loci and targeted non-conserved nucleotides by primer extension to distinguish between template from the two species (Figure 1). Quantitative interspecies qicPCR uses the entire genome of a single chimpanzee as a competitor. This results in the requirement for only a single competitor sample for all assays and dramatically increasing the potential of large numbers of loci to be analysed in multiplex. As the rate of fixed (non-polymorphic) interspecies sequence divergence is ∼1% (26) then >3 × 107 single nucleotides could potentially be targeted. Therefore, qicPCR can potentially be used to analyse the majority of CNVs. These factors, together with accuracy, low cost and potential application to array platforms make it potentially amenable to studies performing multiplex high throughput association analysis of CNVs. Here, we establish proof of concept by first accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of qicPCR to analyse CNVs in association studies by genotyping chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.Figure 1.

Bottom Line: Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility.Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts.In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychological Medicine, Wales School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. williamsnm@cf.ac.uk

ABSTRACT
Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

Show MeSH
Related in: MedlinePlus