Mitochondrial single nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using cleavable biotinylated dideoxynucleotides.

Abstract

Characterization of mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) and mutations is crucial for disease diagnosis, which requires accurate and sensitive detection methods and quantification due to mitochondrial heteroplasmy. We report here the characterization of mutations for myoclonic epilepsy with ragged red fibers syndrome using chemically cleavable biotinylated dideoxynucleotides and a mass spectrometry (MS)-based solid phase capture (SPC) single base extension (SBE) assay. The method effectively eliminates unextended primers and primer dimers, and the presence of cleavable linkers between the base and biotin allows efficient desalting and release of the DNA products from solid phase for MS analysis. This approach is capable of high multiplexing, and the use of different length linkers for each of the purines and each of the pyrimidines permits better discrimination of the four bases by MS. Both homoplasmic and heteroplasmic genotypes were accurately determined on different mtDNA samples. The specificity of the method for mtDNA detection was validated by using mitochondrial DNA-negative cells. The sensitivity of the approach permitted detection of less than 5% mtDNA heteroplasmy levels. This indicates that the SPC-SBE approach based on chemically cleavable biotinylated dideoxynucleotides and MS enables rapid, accurate, and sensitive genotyping of mtDNA and has broad applications for genetic analysis.

• Authors: Chunmei Qiu, Shiv Kumar, Jia Guo, Jiesheng Lu, Shundi Shi, Sergey M Kalachikov, James J Russo, Ali B Naini, Eric A Schon, Jingyue Ju
• Journal: Analytical biochemistry (Anal Biochem) Vol. 427 Issue 2 Pg. 202-10 (Aug 15 2012) ISSN: 1096-0309 [Electronic] United States
• PMID: 22579594 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: Copyright © 2012 Elsevier Inc. All rights reserved.

Chemical References

• DNA, Mitochondrial
• Dideoxynucleotides
• Purines
• Pyrimidines
• Biotin
• Streptavidin

Topics

• Base Sequence
• Biotin (chemistry)
• Biotinylation
• Cell Line
• DNA Fingerprinting (methods)
• DNA, Mitochondrial (analysis)
• Dideoxynucleotides (chemistry, genetics)
• Humans
• MERRF Syndrome (diagnosis, genetics)
• Mitochondria (chemistry, genetics)
• Molecular Sequence Data
• Mutation
• Polymerase Chain Reaction
• Polymorphism, Single Nucleotide
• Purines (chemistry)
• Pyrimidines (chemistry)
• Sensitivity and Specificity
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• Streptavidin (chemistry)