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Official Journal of the Human Genome Organisation

Fig. 2 | Genomic Medicine

Fig. 2

From: Parallel analysis of tetramerization domain mutants of the human p53 protein using PCR colonies

Fig. 2

(A) Polony method used to quantify mutants during p53 growth competition. Plasmid DNA was initially isolated from the culture and used as template in a polony PCR. Common primers amplified all STs, each associated with a different p53 mutant and resulting in an individual polony. Polonies were identified by conducting six sequential single-base extensions using fluorescently-labeled nucleotides. The position of each polony was manually logged after sybr green staining using Metamorph software (Universal Imaging). Data from each extension was assembled into the final code sequence using a simple routine developed in our laboratory. In the representative frames above, two polonies are traced through all six extensions. Upper: tag sequence “AACAAA” corresponds to Gln331Gly. Lower: tag sequence “TCCCAA” corresponds to Gln331Met. (B) Expression plasmids were constructed using the p415CYC1 vector which carries the constitutive CYC1 promoter

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