MALDI-TOF mass spectrometric detection of multiplex single base extended primers. A study of 17 y-chromosome single-nucleotide polymorphisms.
One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.
Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, 11 Frederik V's Vej, DK-2100 Copenhagen Ø, Denmark. jonas.mengel@forensic.ku.dk
J
J
Mengel-Jørgensen
J J
JJ
Sanchez
C
C
Børsting
F
F
Kirpekar
N
N
Morling
D000483:Alleles; D001710:Biotin / Q000737:chemistry; D041322:Chromosomes, Human, Y; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001710:Biotin