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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.

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Representative polony gel for patient 50 following extensions with fluorescently labeled dATP (green) and dGTP (red). This is a composite image generated from the independent extensions with either Cy5-dATP or Cy5-dGTP as described in figure 1. The colors used in the image are artificial.
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Figure 2: Representative polony gel for patient 50 following extensions with fluorescently labeled dATP (green) and dGTP (red). This is a composite image generated from the independent extensions with either Cy5-dATP or Cy5-dGTP as described in figure 1. The colors used in the image are artificial.

Mentions: Polony quantification of relative expression levels was determined to be sensitive enough to detect variation in relative levels of 4208A and 4208G across patients. For example, a polony gel for patient 50 clearly shows elevated expression of PKD2 transcripts bearing the 4208G SNP (Figure 2, red polonies) compared to the 4208A SNP (Figure 2, green polonies). Compared to the previously published data of Yan et al., polony quantification was able to detect the elevated 4208G to 4208A expression levels in the same three patients, namely patients 50, 50-3 and 50-5. Furthermore, similar relative allelic expression levels were detected in all patients using both methods, and the rank order of relative expression levels across patients also remained largely unaffected by the choice of method. It should be noted that analysis of 4208A/G levels in genomic DNA from four of the eight patients was close to the expected 1:1 4208A to 4208G levels (Table 2).


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

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

Representative polony gel for patient 50 following extensions with fluorescently labeled dATP (green) and dGTP (red). This is a composite image generated from the independent extensions with either Cy5-dATP or Cy5-dGTP as described in figure 1. The colors used in the image are artificial.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Representative polony gel for patient 50 following extensions with fluorescently labeled dATP (green) and dGTP (red). This is a composite image generated from the independent extensions with either Cy5-dATP or Cy5-dGTP as described in figure 1. The colors used in the image are artificial.
Mentions: Polony quantification of relative expression levels was determined to be sensitive enough to detect variation in relative levels of 4208A and 4208G across patients. For example, a polony gel for patient 50 clearly shows elevated expression of PKD2 transcripts bearing the 4208G SNP (Figure 2, red polonies) compared to the 4208A SNP (Figure 2, green polonies). Compared to the previously published data of Yan et al., polony quantification was able to detect the elevated 4208G to 4208A expression levels in the same three patients, namely patients 50, 50-3 and 50-5. Furthermore, similar relative allelic expression levels were detected in all patients using both methods, and the rank order of relative expression levels across patients also remained largely unaffected by the choice of method. It should be noted that analysis of 4208A/G levels in genomic DNA from four of the eight patients was close to the expected 1:1 4208A to 4208G levels (Table 2).

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