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Detection of allelic variations of human gene expression by polymerase colonies.

Butz JA, Yan H, Mikkilineni V, Edwards JS - BMC Genet. (2004)

Bottom Line: To validate this technique, the relative expression levels of PKD2 in a family of heterozygous patients bearing the 4208G/A SNP were examined and compared to the literature.We were able to reproduce the results of allelic variation in gene expression using an accurate technology known as polymerase colonies.Therefore, we have demonstrated the utility of this method in human gene expression analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA. butz@che.udel.edu

ABSTRACT

Background: Quantification of variations of human gene expression is complicated by the small differences between different alleles. Recent work has shown that variations do exist in the relative allelic expression levels in certain genes of heterozygous individuals. Herein, we describe the application of an immobilized polymerase chain reaction technique as an alternative approach to measure relative allelic differential expression.

Results: Herein, we report a novel assay, based on immobilized polymerase colonies, that accurately quantifies the relative expression levels of two alleles in a given sample. Mechanistically, this was accomplished by PCR amplifying a gene in a cDNA library in a thin polyacrylamide gel. By immobilizing the PCR, it is ensured that each transcript gives rise to only a single immobilized PCR colony, or "polony". Once polony amplified, the two alleles of the gene were differentially labeled by performing in situ sequencing with fluorescently labeled nucleotides. For these sets of experiments, silent single nucleotide polymorphisms (SNPs) were used to discriminate the two alleles. Finally, a simple count was then performed on the differentially labeled polonies in order to determine the relative expression levels of the two alleles. To validate this technique, the relative expression levels of PKD2 in a family of heterozygous patients bearing the 4208G/A SNP were examined and compared to the literature.

Conclusions: We were able to reproduce the results of allelic variation in gene expression using an accurate technology known as polymerase colonies. Therefore, we have demonstrated the utility of this method in human gene expression analysis.

Show MeSH
Sample size required to differentiate various proportions of allelic expression. The sample size is shown that is required to differentiate various levels of allelic variation of gene expression. We have required a power of 0.90 for these data.
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Figure 3: Sample size required to differentiate various proportions of allelic expression. The sample size is shown that is required to differentiate various levels of allelic variation of gene expression. We have required a power of 0.90 for these data.

Mentions: There are at least two major advantages to the polony approach for measuring allelic variation in gene expression. First, the measurements are digital and hence more accurate than the 'analog' approaches for quantifying allelic variation (see Figure 3). In addition, the polony approach for measuring allelic variation provides an absolute measurement of the relative expression of the two alleles, whereas other approaches are normalized to the population average which is assumed to be 1:1.


Detection of allelic variations of human gene expression by polymerase colonies.

Butz JA, Yan H, Mikkilineni V, Edwards JS - BMC Genet. (2004)

Sample size required to differentiate various proportions of allelic expression. The sample size is shown that is required to differentiate various levels of allelic variation of gene expression. We have required a power of 0.90 for these data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC373445&req=5

Figure 3: Sample size required to differentiate various proportions of allelic expression. The sample size is shown that is required to differentiate various levels of allelic variation of gene expression. We have required a power of 0.90 for these data.
Mentions: There are at least two major advantages to the polony approach for measuring allelic variation in gene expression. First, the measurements are digital and hence more accurate than the 'analog' approaches for quantifying allelic variation (see Figure 3). In addition, the polony approach for measuring allelic variation provides an absolute measurement of the relative expression of the two alleles, whereas other approaches are normalized to the population average which is assumed to be 1:1.

Bottom Line: To validate this technique, the relative expression levels of PKD2 in a family of heterozygous patients bearing the 4208G/A SNP were examined and compared to the literature.We were able to reproduce the results of allelic variation in gene expression using an accurate technology known as polymerase colonies.Therefore, we have demonstrated the utility of this method in human gene expression analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA. butz@che.udel.edu

ABSTRACT

Background: Quantification of variations of human gene expression is complicated by the small differences between different alleles. Recent work has shown that variations do exist in the relative allelic expression levels in certain genes of heterozygous individuals. Herein, we describe the application of an immobilized polymerase chain reaction technique as an alternative approach to measure relative allelic differential expression.

Results: Herein, we report a novel assay, based on immobilized polymerase colonies, that accurately quantifies the relative expression levels of two alleles in a given sample. Mechanistically, this was accomplished by PCR amplifying a gene in a cDNA library in a thin polyacrylamide gel. By immobilizing the PCR, it is ensured that each transcript gives rise to only a single immobilized PCR colony, or "polony". Once polony amplified, the two alleles of the gene were differentially labeled by performing in situ sequencing with fluorescently labeled nucleotides. For these sets of experiments, silent single nucleotide polymorphisms (SNPs) were used to discriminate the two alleles. Finally, a simple count was then performed on the differentially labeled polonies in order to determine the relative expression levels of the two alleles. To validate this technique, the relative expression levels of PKD2 in a family of heterozygous patients bearing the 4208G/A SNP were examined and compared to the literature.

Conclusions: We were able to reproduce the results of allelic variation in gene expression using an accurate technology known as polymerase colonies. Therefore, we have demonstrated the utility of this method in human gene expression analysis.

Show MeSH