Affinity methods for purification of DNA sequencing reaction products for mass spectrometric analysis.
Time-of-flight mass spectrometry has the potential to replace gel electrophoresis for rapid and accurate analysis of DNA sequencing mixtures. However, impurities in the Sanger sequencing reaction solutions can complicate and degrade the mass spectrometric performance. Therefore, a fast purification procedure is necessary for mass spectrometric analysis. Two affinity methods were tested for the capture of the target fragments: a probe strand, complementary to the primer used to initiate synthesis of the Sanger fragments, is immobilized to a solid support either before or after hybridization so that the impurities are readily separated by filtering and washing the support material. The approaches were tested using mock sequencing mixtures assembled from synthetic DNA strands, to which representative impurities could be added. The results showed that the latter method has better overall yield. The recovered fragments were analyzed by matrix-assisted laser desorption/ionization.
Chou
C W
CW
Department of Chemistry and Biochemistry, Arizona State University, Tempe 85287-1604, USA.
Bingham
S E
SE
Williams
P
P
D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D002846:Chromatography, Affinity; D004247:DNA / Q000302:isolation & purification*; D015342:DNA Probes / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin
D001426:Bacterial Proteins; D015342:DNA Probes; D004247:DNA; D019809:Streptavidin