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Genotyping SNPs Using a UV Photocleavable Oligonucleotide in MALDI-TOF MS

Published

Author(s)

Peter M. Vallone, K Fahr, M Kostrzewa

Abstract

Matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) coupled with allele specific primer extension is a proven method for typing single nucleotide polymorphisms (SNPs). A novel modification upon this methodology is the incorporation of a photocleavable linker within the extension primer. After completion of the primer extension reaction photocleavage of the extension products results in two DNA fragments of lower mass. Typically, the smaller cleavage product, which contains the genotyping information is in the range of 1000 daltons to 3000 daltons. The decrease in primer mass allows for higher sensitivity in mass spectrometric measurement and increases the potential for higher levels of multiplexing. Mass spectrometric analysis disturbing peaks due to salt adducts and doubly charged ions are diminished when analyzing lower mass DNA fragments. Here we illustrate the methodology for using photocleavable modified extension primers for detection of SNPs located on the Y chromosome. Genomic templates were prepared from anonymous male donors. Five regions of the Y chromosome containing the SNP markers M9, M42, M45, M89 and M96 were amplified by PCR, treated with shrimp alkaline phosphatase, and subjected to primer extension reactions using primers containing a photocleavable building block at specific sites. Post elongation, the extension primers were desalted and subjected to UV irradiation to cleave the products at the photocleavable site. Subsequently, the small fragments derived from the 3' ends of the molecules containing the genotype information were analyzed by MALDI-TOF MS using a 3-hydroxypicolinic acid matrix.
Citation
Methods in Molecular Biology
Volume
297

Keywords

geneotyping, multiplex, photocleavable, primer extension, SNP

Citation

Vallone, P. , Fahr, K. and Kostrzewa, M. (2005), Genotyping SNPs Using a UV Photocleavable Oligonucleotide in MALDI-TOF MS, Methods in Molecular Biology (Accessed April 22, 2024)
Created March 17, 2005, Updated February 17, 2017