Recovery of nucleic acids from immobilized biotin-streptavidin complexes using ammonium hydroxide and applications in MALDI-TOF mass spectrometry.
The effects of ammonium hydroxide treatment of complexes consisting of biotinylated nucleic acids and immobilized streptavidin were investigated. It was found that incubation of such complexes with ammonium hydroxide at room temperature leads to denaturation of double-stranded DNA molecules, liberating only the complementary nonbiotinylated strand, whereas incubation at elevated temperatures leads to an efficient dissociation of biotin-streptavidin complexes. The introduced procedure is especially suitable as a purification and conditioning format prior to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of DNA from complex enzymatic reactions. This is demonstrated by analysis of polymerase chain reaction (PCR) and sequencing products.
Jurinke
C
C
Department of Biochemistry and Molecular Biology, University of Hamburg, Germany.
van den Boom
D
D
Collazo
V
V
Lüchow
A
A
Jacob
A
A
Köster
H
H
D064753:Ammonium Hydroxide; D001426:Bacterial Proteins; D001710:Biotin; D004247:DNA / Q000737:chemistry; D006878:Hydroxides; D009696:Nucleic Acids / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019809:Streptavidin
D001426:Bacterial Proteins; D006878:Hydroxides; D009696:Nucleic Acids; D064753:Ammonium Hydroxide; D001710:Biotin; D004247:DNA; D019809:Streptavidin