Analysis of ligase chain reaction products via matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry.
A rapid and accurate detection of ligation products generated in ligase chain reactions (LCR) by using matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) is reported. LCR with Pfu DNA ligase was performed with a wild-type template and a template carrying a single point mutation within the Escherichia coli lacI gene as a model system. Starting from about 1 fmol of template DNA the ligation product generated in the positive reactions was analyzed with HPLC and MALDI-TOF-MS, whereby the need of proper sample purification prior to mass spectrometric analysis was demonstrated. A purification procedure with a high potential for automation using streptavidin-coated magnetic particles and ultrafiltration was introduced. Plasmid DNA and short single-stranded oligonucleotides have been used as template. A point mutation could be discriminated from the wild-type template due to the absence or presence of ligation product. This approach allows the rapid-specific detection of template DNA in femtomole amounts and moreover can distinguish between sequence variations in DNA molecules down to point mutations without the need for labeling, gel electrophoresis, membrane transfer, or hybridization procedures.
Jurinke
C
C
Department of Biochemistry, Faculty of Chemistry, University of Hamburg, D-20146, Germany.
van den Boom
D
D
Jacob
A
A
Tang
K
K
Wörl
R
R
Köster
H
H
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D011088:DNA Ligases; D004252:DNA Mutational Analysis / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D007763:Lac Operon; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011088:DNA Ligases