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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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Determining relative allelic expression using polony technology. (A) Initially, the gene of interest is polony amplified from cDNA. The primers are designed to amplify the region of the gene bearing a silent SNP, which is used to discriminate the two alleles. In this illustration, one allele bears an A-T pairing at a particular position whereas the other allele has a G-C pairing instead. Note: one of primers has a 5' acrydite modification (star), which is eventually required for distinguishing these two alleles. (B) Following polony amplification, the gel is stained with SybrGreenI and scanned with a microarray scanner in order to ensure that the polony amplification worked properly. Each black dot represents the polony amplification of one copy of the desired first-strand cDNA template. (C) Prior to hybridization of sequencing primer, the polony is made single stranded by denaturing the double stranded in formamide followed by the removal of non-acrydited strand using electrophoresis. (D) A sequencing primer is hybridized to the single stranded polony, a single base extension is performed with Cy5-dATP and then the gel is scanned. To label the other allele, the process of denaturation, hybridation, extension, and scanning is repeated, but the extension is performed with Cy5-dGTP instead. (E) To determine the relative expression levels of the two alleles, a composite image from the Cy5-dATP and Cy5-dGTP extensions is first generated and then the number of polonies (transcripts) for each of the two alleles is counted. In this particular example, the "G-C" SNP represents 62.5% of the population (i.e., 5 out of 8 polonies). Figure adapted from Yan et al [6].
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Figure 1: Determining relative allelic expression using polony technology. (A) Initially, the gene of interest is polony amplified from cDNA. The primers are designed to amplify the region of the gene bearing a silent SNP, which is used to discriminate the two alleles. In this illustration, one allele bears an A-T pairing at a particular position whereas the other allele has a G-C pairing instead. Note: one of primers has a 5' acrydite modification (star), which is eventually required for distinguishing these two alleles. (B) Following polony amplification, the gel is stained with SybrGreenI and scanned with a microarray scanner in order to ensure that the polony amplification worked properly. Each black dot represents the polony amplification of one copy of the desired first-strand cDNA template. (C) Prior to hybridization of sequencing primer, the polony is made single stranded by denaturing the double stranded in formamide followed by the removal of non-acrydited strand using electrophoresis. (D) A sequencing primer is hybridized to the single stranded polony, a single base extension is performed with Cy5-dATP and then the gel is scanned. To label the other allele, the process of denaturation, hybridation, extension, and scanning is repeated, but the extension is performed with Cy5-dGTP instead. (E) To determine the relative expression levels of the two alleles, a composite image from the Cy5-dATP and Cy5-dGTP extensions is first generated and then the number of polonies (transcripts) for each of the two alleles is counted. In this particular example, the "G-C" SNP represents 62.5% of the population (i.e., 5 out of 8 polonies). Figure adapted from Yan et al [6].

Mentions: Recent work has shown that variations exist in the relative allelic expression levels in certain genes of heterozygous individuals [6]. Herein, we describe the application of polony technology as an alternative approach to measure relative allelic differential expression. Initially, the two alleles for a particular gene were polony amplified with primers which flank the SNP (Figure 1A), giving rise to one polony per transcript. The polonies were observed after the gel was stained with SyBr Green I and scanned with a microarray scanner (Figure 1B). An additional step was required to discriminate the two alleles which involved: 1) making the polony single stranded by formamide denaturation and electrophoresis (Figure 1C); 2) hybridization of a sequencing primer with the 3' end preceding the SNP; 3) a single-base extension with fluorescent nucleotides to differentially label the two alleles (Figure 1D); and 4) scanning the gel to determine allelic expression levels (Figure 1E). This approach was able to successfully identify variations in the 4208A/G alleles of PKD2 in heterozygous patients as previous described [6].


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

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

Determining relative allelic expression using polony technology. (A) Initially, the gene of interest is polony amplified from cDNA. The primers are designed to amplify the region of the gene bearing a silent SNP, which is used to discriminate the two alleles. In this illustration, one allele bears an A-T pairing at a particular position whereas the other allele has a G-C pairing instead. Note: one of primers has a 5' acrydite modification (star), which is eventually required for distinguishing these two alleles. (B) Following polony amplification, the gel is stained with SybrGreenI and scanned with a microarray scanner in order to ensure that the polony amplification worked properly. Each black dot represents the polony amplification of one copy of the desired first-strand cDNA template. (C) Prior to hybridization of sequencing primer, the polony is made single stranded by denaturing the double stranded in formamide followed by the removal of non-acrydited strand using electrophoresis. (D) A sequencing primer is hybridized to the single stranded polony, a single base extension is performed with Cy5-dATP and then the gel is scanned. To label the other allele, the process of denaturation, hybridation, extension, and scanning is repeated, but the extension is performed with Cy5-dGTP instead. (E) To determine the relative expression levels of the two alleles, a composite image from the Cy5-dATP and Cy5-dGTP extensions is first generated and then the number of polonies (transcripts) for each of the two alleles is counted. In this particular example, the "G-C" SNP represents 62.5% of the population (i.e., 5 out of 8 polonies). Figure adapted from Yan et al [6].
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Related In: Results  -  Collection

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Figure 1: Determining relative allelic expression using polony technology. (A) Initially, the gene of interest is polony amplified from cDNA. The primers are designed to amplify the region of the gene bearing a silent SNP, which is used to discriminate the two alleles. In this illustration, one allele bears an A-T pairing at a particular position whereas the other allele has a G-C pairing instead. Note: one of primers has a 5' acrydite modification (star), which is eventually required for distinguishing these two alleles. (B) Following polony amplification, the gel is stained with SybrGreenI and scanned with a microarray scanner in order to ensure that the polony amplification worked properly. Each black dot represents the polony amplification of one copy of the desired first-strand cDNA template. (C) Prior to hybridization of sequencing primer, the polony is made single stranded by denaturing the double stranded in formamide followed by the removal of non-acrydited strand using electrophoresis. (D) A sequencing primer is hybridized to the single stranded polony, a single base extension is performed with Cy5-dATP and then the gel is scanned. To label the other allele, the process of denaturation, hybridation, extension, and scanning is repeated, but the extension is performed with Cy5-dGTP instead. (E) To determine the relative expression levels of the two alleles, a composite image from the Cy5-dATP and Cy5-dGTP extensions is first generated and then the number of polonies (transcripts) for each of the two alleles is counted. In this particular example, the "G-C" SNP represents 62.5% of the population (i.e., 5 out of 8 polonies). Figure adapted from Yan et al [6].
Mentions: Recent work has shown that variations exist in the relative allelic expression levels in certain genes of heterozygous individuals [6]. Herein, we describe the application of polony technology as an alternative approach to measure relative allelic differential expression. Initially, the two alleles for a particular gene were polony amplified with primers which flank the SNP (Figure 1A), giving rise to one polony per transcript. The polonies were observed after the gel was stained with SyBr Green I and scanned with a microarray scanner (Figure 1B). An additional step was required to discriminate the two alleles which involved: 1) making the polony single stranded by formamide denaturation and electrophoresis (Figure 1C); 2) hybridization of a sequencing primer with the 3' end preceding the SNP; 3) a single-base extension with fluorescent nucleotides to differentially label the two alleles (Figure 1D); and 4) scanning the gel to determine allelic expression levels (Figure 1E). This approach was able to successfully identify variations in the 4208A/G alleles of PKD2 in heterozygous patients as previous described [6].

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