Stable-isotope-assisted MALDI-TOF mass spectrometry for accurate determination of nucleotide compositions of PCR products.
In parallel with a large-scale sequencing effort, the human genome project will need next-generation tools for accurate and efficient analyses of the enormous pool of DNA sequences. Such analyses are required for (a) validation of DNA sequences, (b) comparison of a parent (known) sequence with a related (unknown) sequence, and (c) characterization of sequence polymorphisms in various genes, especially those associated with genetically inherited human diseases. Here, we report a novel method that combines stable isotope 13C/15N labeling of PCR products of the target sequences with analysis of the mass shifts by mass spectrometry (MS). The mass shift due to the labeling of a single type of nucleotide (i.e., A, T, G, or C) reveals the number of that type of nucleotide in a given DNA fragment. Using this technique, we have accurately determined nucleotide compositions of DNA fragments. The method has also been applied to score a known single-nucleotide polymorphism (SNP). The comparisons of nucleotide compositions determined by our method among homologous sequences are useful in sequence validation, sequence comparison, and characterizations of sequence polymorphisms.
Chen
X
X
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA. xchen@telomere.lanl.gov
Fei
Z
Z
Smith
L M
LM
Bradbury
E M
EM
Majidi
V
V
D015820:Cadherins / Q000235:genetics; D002247:Carbon Isotopes; D006801:Humans; D009587:Nitrogen Isotopes; D009711:Nucleotides / Q000032:analysis*; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D015820:Cadherins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D009711:Nucleotides; D009841:Oligonucleotides