Parallel analysis of mutant human glucose 6-phosphate dehydrogenase in yeast using PCR colonies

We demonstrate a highly parallel strategy to analyze the impact of single nucleotide mutations on protein function. Using our method, it is possible to screen a population and quickly identify a subset of functionally interesting mutants. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools, and polymerase colonies. A defined mutant human glucose-6-phosphate-dehydrogenase library was constructed which contains all possible single nucleotide missense mutations in the eight-residue glucose-6-phosphate binding peptide of the enzyme. Mutant human enzymes were expressed in a zwf1 (gene encoding yeast homologue) deletion strain of Saccharomyces cerevisiae. Growth rates of the 54 mutant strains arising from this library were measured in parallel in conditions selective for active hG6PD. Several residues were identified which tolerated no mutations (Asp200, His201 and Lys205) and two (Ile199 and Leu203) tolerated several substitutions. Arg198, Tyr202, and Gly204 tolerated only 1-2 specific substitutions. Generalizing from the positions of tolerated and non-tolerated amino acid substitutions, hypotheses were generated about the functional role of specific residues, which could, potentially, be tested using higher resolution/lower throughput methods.     

Use of nanocolonies to detect minimal residual disease in patients with leukemia t(8;12)

Molecular Biology - A complete diagnostic procedure was developed to detect single molecules of the AML1-ETO mRNA in whole blood and bone marrow samples of leukemia t(8;21)(q22;q22) patients. The...     

Scientific and practical applications of molecular colonies

The review briefs the history of the invention of the molecular colony techique, also known as a polony technology; applications of this method to studies of the reactions between single RNA molecules, ultrasensitive diagnosis, gene cloning, and in vitro screening, as well as the concepts of the origin of life that regard molecular colonies as a prototype of living organisms.     

Nanocolonies: Detection, cloning, and analysis of individual molecules

Nanocolonies (other names molecular colonies or polonies) are formed upon template nanomolecule (DNA or RNA) amplification in immobilized medium with efficient pore size in the nanometer range. This work deals with the principle, invention, development, and diverse nanocolony applications based on their unique abilities to compartmentalize amplification and expression of individual DNA and RNA molecules, including studying reactions between single molecules, digital molecular diagnostics, in vitro gene cloning and expression, as well as identification of the molecular cis-elements including DNA sequencing, analysis of single-nucleotide polymorphism, and alternative splicing investigation.