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].