MALDI-TOF mass spectrometric detection of multiplex single base extended primers: A study of 17 y-chromosome single-nucleotide polymorphisms

TY - JOUR

T1 - MALDI-TOF mass spectrometric detection of multiplex single base extended primers

T2 - A study of 17 y-chromosome single-nucleotide polymorphisms

AU - Mengel-From, Jonas

AU - Sanchez Sanchez, Juan Jose

AU - Børsting, Claus

AU - Kirpekar, Finn

AU - Morling, N

N1 - Keywords: Alleles; Biotin; Chromosomes, Human, Y; Humans; Polymorphism, Single Nucleotide; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

U2 - 10.1021/ac049264k

DO - 10.1021/ac049264k

M3 - Journal article

C2 - 15481951

SN - 0003-2700

VL - 76

SP - 6039

EP - 6045

JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 20

ER -