Abstract
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.
Highlights
Human mitochondrial DNA is a circular genome with 16569 base pairs, and exists as hundreds to thousands of copies in the mitochondrial matrix
Our results suggest that the relative sensitivity of the SURVEYOR Nuclease (SN)/WAVE-HS platform for the detection of mitochondrial DNA (mtDNA) heteroplasmy is marginally higher than that of denaturing high-performance liquid chromatography (DHPLC) for one single nucleotide polymorphism (SNP), both techniques determine heteroplasmy qualitatively
We have provided a practical approach for the screening and evaluation of the potential interference of nuclear mitochondrial DNA sequences (NUMTs) on mtDNA detection during DHPLC and SN/WAVE-HS analysis from primers by using published primer sets as an example
Summary
Human mitochondrial DNA (mtDNA) is a circular genome with 16569 base pairs (bp), and exists as hundreds to thousands of copies in the mitochondrial matrix. PSQ is a powerful technique for the quantification of mtDNA heteroplasmy for a known single nucleotide polymorphism (SNP) or mutation without the need of establishing a standard curve [4,5], whereas allelerefractory mutation system (AMRS)-based quantitative polymerase chain reaction (qPCR) represents a highly sensitive technique that requires a standard curve made by mixing 2 DNA samples containing different alleles [6] These kinds of methods are not suitable for the search on unknown variants or mutations, which are often present at low-level heteroplasmy as well, over entire mtDNA in various studies. DHPLC represents a kind of sensitive and high-throughput, qualitative, technique to screen heteroplasmic variants for entire mtDNA [7,8]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
