Enzymatic strategies for the characterization of nucleic acids by electrospray ionization mass spectrometry.
Electrospray ionization mass spectrometry (ESI-MS) is a powerful technique used for the identification and characterization of DNA polymorphisms. Continual improvement in instrument design assures high mass measurement accuracy, sensitivity, and resolving power. This work describes an eclectic array of enzymatic strategies we have invoked in order to detect single-nucleotide polymorphisms by ESI-MS, although other applications may be envisioned. One strategy combines the use of two enzymes, exonuclease III and lambda exonuclease, to provide a ladder of single-stranded DNA fragments for straightforward sequence identification by mass spectrometry. A second strategy combines restriction enzymes to screen for polymorphisms present within specific amplicons. Finally, we describe the use of stable-isotope-labeled nucleotides for the determination of length and base composition of a PCR product.
Null
Allison P
AP
W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Proteomics Research Center, and Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Benson
Linda M
LM
Muddiman
David C
DC
D005090:Exodeoxyribonucleases / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D009696:Nucleic Acids / Q000032:analysis / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014764:Viral Proteins
D009696:Nucleic Acids; D009841:Oligonucleotides; D014764:Viral Proteins; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C043586:exo protein, Bacteriophage lambda