Single-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.

It is thought that changes in mitochondrial DNA are associated with many degenerative diseases, including Alzheimer's and diabetes. Much of the evidence, however, depends on correlating disease states with changing levels of heteroplasmy within populations of mitochondrial genomes, rather than...

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Autores principales:	Adam Osborne, Arthur H Reis, Loren Bach, Lawrence J Wangh
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2009
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bf81dd411db7464f853d09c54cfe91f2
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spelling	oai:doaj.org-article:bf81dd411db7464f853d09c54cfe91f22021-11-25T06:22:35ZSingle-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.1932-620310.1371/journal.pone.0005636https://doaj.org/article/bf81dd411db7464f853d09c54cfe91f22009-05-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19461959/?tool=EBIhttps://doaj.org/toc/1932-6203It is thought that changes in mitochondrial DNA are associated with many degenerative diseases, including Alzheimer's and diabetes. Much of the evidence, however, depends on correlating disease states with changing levels of heteroplasmy within populations of mitochondrial genomes, rather than individual mitochondrial genomes. Thus these measurements are likely to either overestimate the extent of heteroplasmy due to technical artifacts, or underestimate the actual level of heteroplasmy because only the most abundant changes are observable. In contrast, Single Molecule (SM) LATE-PCR analysis achieves efficient amplification of single-stranded amplicons from single target molecules. The product molecules, in turn, can be accurately sequenced using a convenient Dilute-'N'-Go protocol, as shown here. Using these novel technologies we have rigorously analyzed levels of mitochondrial genome heteroplasmy found in single hair shafts of healthy adult individuals. Two of the single molecule sequences (7% of the samples) were found to contain mutations. Most of the mtDNA sequence changes, however, were due to the presence of laboratory contaminants. Amplification and sequencing errors did not result in mis-identification of mutations. We conclude that SM-LATE-PCR in combination with Dilute-'N'-Go Sequencing are convenient technologies for detecting infrequent mutations in mitochondrial genomes, provided great care is taken to control and document contamination. We plan to use these technologies in the future to look for age, drug, and disease related mitochondrial genome changes in model systems and clinical samples.Adam OsborneArthur H ReisLoren BachLawrence J WanghPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 4, Iss 5, p e5636 (2009)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Adam Osborne Arthur H Reis Loren Bach Lawrence J Wangh Single-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.
description	It is thought that changes in mitochondrial DNA are associated with many degenerative diseases, including Alzheimer's and diabetes. Much of the evidence, however, depends on correlating disease states with changing levels of heteroplasmy within populations of mitochondrial genomes, rather than individual mitochondrial genomes. Thus these measurements are likely to either overestimate the extent of heteroplasmy due to technical artifacts, or underestimate the actual level of heteroplasmy because only the most abundant changes are observable. In contrast, Single Molecule (SM) LATE-PCR analysis achieves efficient amplification of single-stranded amplicons from single target molecules. The product molecules, in turn, can be accurately sequenced using a convenient Dilute-'N'-Go protocol, as shown here. Using these novel technologies we have rigorously analyzed levels of mitochondrial genome heteroplasmy found in single hair shafts of healthy adult individuals. Two of the single molecule sequences (7% of the samples) were found to contain mutations. Most of the mtDNA sequence changes, however, were due to the presence of laboratory contaminants. Amplification and sequencing errors did not result in mis-identification of mutations. We conclude that SM-LATE-PCR in combination with Dilute-'N'-Go Sequencing are convenient technologies for detecting infrequent mutations in mitochondrial genomes, provided great care is taken to control and document contamination. We plan to use these technologies in the future to look for age, drug, and disease related mitochondrial genome changes in model systems and clinical samples.
format	article
author	Adam Osborne Arthur H Reis Loren Bach Lawrence J Wangh
author_facet	Adam Osborne Arthur H Reis Loren Bach Lawrence J Wangh
author_sort	Adam Osborne
title	Single-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.
title_short	Single-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.
title_full	Single-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.
title_fullStr	Single-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.
title_full_unstemmed	Single-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.
title_sort	single-molecule late-pcr analysis of human mitochondrial genomic sequence variations.
publisher	Public Library of Science (PLoS)
publishDate	2009
url	https://doaj.org/article/bf81dd411db7464f853d09c54cfe91f2
work_keys_str_mv	AT adamosborne singlemoleculelatepcranalysisofhumanmitochondrialgenomicsequencevariations AT arthurhreis singlemoleculelatepcranalysisofhumanmitochondrialgenomicsequencevariations AT lorenbach singlemoleculelatepcranalysisofhumanmitochondrialgenomicsequencevariations AT lawrencejwangh singlemoleculelatepcranalysisofhumanmitochondrialgenomicsequencevariations
_version_	1718413824451674112

Single-molecule LATE-PCR analysis of human mitochondrial genomic sequence variations.

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