Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.

High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR...

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Autores principales:	Hsiu-Chuan Yen, Shiue-Li Li, Wei-Chien Hsu, Petrus Tang
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9143dd89883a4c719cca49b2d6e22f38
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spelling	oai:doaj.org-article:9143dd89883a4c719cca49b2d6e22f382021-11-18T08:26:27ZInterference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.1932-620310.1371/journal.pone.0092817https://doaj.org/article/9143dd89883a4c719cca49b2d6e22f382014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24664244/?tool=EBIhttps://doaj.org/toc/1932-6203High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detecting human mtDNA variants and found that its performance was slightly better than that of denaturing high-performance liquid chromatography (DHPLC). The potential interference from co-amplified NUMTs on screening mtDNA heteroplasmy when using these 2 highly sensitive techniques was further examined by using 2 published primer sets containing a total of 65 primer pairs, which were originally designed to be used with one of the 2 techniques. We confirmed that 24 primer pairs could amplify NUMTs by conducting bioinformatic analysis and PCR with the DNA from 143B-ρ0 cells. Using mtDNA extracted from the mitochondria of human 143B cells and a cybrid line with the nuclear background of 143B-ρ0 cells, we demonstrated that NUMTs could affect the patterns of chromatograms for cell DNA during SN-WAVE/HS analysis of mtDNA, leading to incorrect judgment of mtDNA homoplasmy or heteroplasmy status. However, we observed such interference only in 2 of 24 primer pairs selected, and did not observe such effects during DHPLC analysis. These results indicate that NUMTs can affect the screening of low-level mtDNA variants, but it might not be predicted by bioinformatic analysis or the amplification of DNA from 143B-ρ0 cells. Therefore, using purified mtDNA from cultured cells with proven purity to evaluate the effects of NUMTs from a primer pair on mtDNA detection by using PCR-based high-sensitivity methods prior to the use of a primer pair in real studies would be a more practical strategy.Hsiu-Chuan YenShiue-Li LiWei-Chien HsuPetrus TangPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 3, p e92817 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Hsiu-Chuan Yen Shiue-Li Li Wei-Chien Hsu Petrus Tang Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.
description	High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detecting human mtDNA variants and found that its performance was slightly better than that of denaturing high-performance liquid chromatography (DHPLC). The potential interference from co-amplified NUMTs on screening mtDNA heteroplasmy when using these 2 highly sensitive techniques was further examined by using 2 published primer sets containing a total of 65 primer pairs, which were originally designed to be used with one of the 2 techniques. We confirmed that 24 primer pairs could amplify NUMTs by conducting bioinformatic analysis and PCR with the DNA from 143B-ρ0 cells. Using mtDNA extracted from the mitochondria of human 143B cells and a cybrid line with the nuclear background of 143B-ρ0 cells, we demonstrated that NUMTs could affect the patterns of chromatograms for cell DNA during SN-WAVE/HS analysis of mtDNA, leading to incorrect judgment of mtDNA homoplasmy or heteroplasmy status. However, we observed such interference only in 2 of 24 primer pairs selected, and did not observe such effects during DHPLC analysis. These results indicate that NUMTs can affect the screening of low-level mtDNA variants, but it might not be predicted by bioinformatic analysis or the amplification of DNA from 143B-ρ0 cells. Therefore, using purified mtDNA from cultured cells with proven purity to evaluate the effects of NUMTs from a primer pair on mtDNA detection by using PCR-based high-sensitivity methods prior to the use of a primer pair in real studies would be a more practical strategy.
format	article
author	Hsiu-Chuan Yen Shiue-Li Li Wei-Chien Hsu Petrus Tang
author_facet	Hsiu-Chuan Yen Shiue-Li Li Wei-Chien Hsu Petrus Tang
author_sort	Hsiu-Chuan Yen
title	Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.
title_short	Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.
title_full	Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.
title_fullStr	Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.
title_full_unstemmed	Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.
title_sort	interference of co-amplified nuclear mitochondrial dna sequences on the determination of human mtdna heteroplasmy by using the surveyor nuclease and the wave hs system.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/9143dd89883a4c719cca49b2d6e22f38
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Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system.

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