What are the currently known reasons for an assay FAILURE?
• Failed amplification reaction • Over-amplified sample Reason: The AcycloPrime reaction uses an excess of SNP primer over dye-AcycloTerminators and thus once the “correct” terminators are used up the DNA polymerase will attempt to incorporate mis-matched bases. • Inadequate sample cleanup after amplification Reason: It is critical that both the excess primers and dNTPs are removed from the reaction using Exo and SAP before performing the AcycloPrime step, remaining primers result in “false” sites being interrogated and dNTPs permit elongation longer than the single base addition desired. • Over-cycling of the AcycloPrime reaction Reason: see explanation above for “Over Amplified sample”. It is possible to perform stepwise cycling, e.g., 5-10 cycles then read FP, then repeat cycling if necessary. • No polarization detected Reason: instrument problem, filter problem, one or more of above? Check to see if allele-specific failure, e.g., some wells positive and others negative or this is a
