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Updated: May 31, 2026

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
An allelic ladder based upon reference alleles for mtDNA SNP analysis using the SNaPshot technique
Stephan Köhnemann1, Petra Pennekamp, Heidi Pfeiffer
1Institute of Legal Medicine, Münster, Germany. stephan.koehnemann@ukmuenster.de
Abstract:
The analysis of mitochondrial DNA (mtDNA) single-nucleotide polymorphisms (SNPs) using the SNaPshot technique (Applied Biosystems) is a fast and sensitive method for the reliable identification of disease-associated mtDNA SNPs, genetic ancestry mtDNA SNPs and forensically important mtDNA SNPs. The detection of many SNPs in one multiplex PCR and one subsequent multiplex minisequencing reaction is challenging for laboratories who want to establish this technique, due to the problem that there is no allelic ladder available for mtDNA SNP analysis via SNaPshot technique. Normally, the laboratory has to invent long-term testing and studies. The interpretation of false and correct alleles is up to some specialists knowing the expected and the estimated size of each allele SNP. We here present a protocol to assemble up to 84 alleles of 42 different mtDNA SNPs in an allelic ladder that is based upon reference alleles. We recommend using allelic ladders/reference alleles for SNP analysis to maintain high-quality analysis standards.
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