A strategy for examining complex mixtures of deoxyoligonucleotides using ion-pair-reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and informatics.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ion-pair-reverse-phase high-performance liquid chromatography (IP-RP HPLC) techniques were combined to determine the sequence identity of short single-stranded deoxyoligonucleotides. This methodology is demonstrated using a commercially available multiplex set of eight primer pairs. The primer pairs were separated and collected by IP-RP HPLC. Partial sequence information for IP-RP HPLC fractions was obtained from analyzing exonuclease digestion products by MALDI-TOF MS. IP-RP HPLC, MALDI-TOF MS, exonuclease digests, and a simple computational algorithm provide an integrated strategy for determining the sequence of short nucleic acid oligomers.
Vallone
Peter M
PM
Biotechnology Division, National Institute of Standards and Technology, 100 Bureau Drive, Mail Stop 8311, Gaithersburg, MD 20899, USA. peter.vallone@nist.gov
Devaney
Joseph M
JM
Marino
Michael A
MA
Butler
John M
JM
D000465:Algorithms; D001699:Biometry / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V