AFFINITY METHODS FOR PURIFICATION OF DNA-SEQUENCING REACTION-PRODUCTSFOR MASS-SPECTROMETRIC ANALYSIS

Time-of-flight mass spectrometry has the potential to replace gel elec trophoresis for rapid and accurate analysis of DNA sequencing mixtures . However impurities in the Sanger sequencing reaction solutions can c omplicate and degrade the mass spectrometric performance. Therefore, a fast purification procedure is necessary for mass spectrometric analy sis. Two affinity methods were tested for the capture of the target fr agments: a probe strand, complementary to the primer used to initiate synthesis of the Sanger fragments, is immobilized to a solid support e ither before or after hybridization so that the impurities are readily separated by filtering and washing the support material. The approach es were tested using mock sequencing mixtures assembled from synthetic DNA strands, to which representative impurities could be added. The r esults showed that the latter method has better overall yield. The rec overed fragments were analyzed by matrix-assisted laser desorption/ion ization.