Optimization of Primer Extension : Guanine Distribution and Single Cycle Reaction

Multiplexing Asymmetric Primer Extension is a complex and lengthy reaction that can be affected by many variables. It is used by AutoGenomics Inc. to help determine patient’s genetic mutations that could affect their course of treatment and to determine particular strains of infective agents. Further optimizing these types of assays could greatly reduce the time to first result which is crucial for the treatment of many diseases. When conducting an assay optimization , an AutoGenomics scientist had an issue with an amplicons and had determined that a G-quadruplex had effected the label incorporation step of the assay . Factors such as target concentration, annealing temperature and cation concentrations were investigated in order to overcome the G-quadruplex. It was determines that the inhibition of the reaction could be best overcome by adding additional MgCl2 to the reaction. In a second optimization project, it was also found that a reaction time of five minutes, reduced from 90 min ,with one cycle was enough to get usable RFUs for all Taq Polymerases .In conclusion, these projects demonstrated ways to improve the performance of some of the Autogenomics assays and to allow for quicker results vital for patients to get the treatment they need.