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Detection and Quantification of Large Scale Deletions of Human Mitochondrial DNA by Real‐time PCR
Author(s) -
Qian Edan,
Xue Julie,
Guo James M.,
Gao Xueshan,
Eaton John W,
Qian Mingwei
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.508.9
Subject(s) - amplicon , mitochondrial dna , biology , microbiology and biotechnology , polymerase chain reaction , dna , real time polymerase chain reaction , cheek , genetics , gene , ecology
Large deletions of mitochondrial DNA (mtDNA) occur in mitochondrial disease, oxidative stress and aging. Three approaches were developed to identify and quantify the deletions in nine cell lines and cheek cells collected from 18 human volunteers.1) mtDNA samples were screened by qPCR (SYBR Green) with over 150 pairs of primers with expected amplicon sizes >1kb. The qPCR conditions were set to amplify short sequences only (<400 bp). 2) Additional primers flanking the possible deletion sequences were designed to pinpoint the deletion sites. 3) The deletions were normalized with two short amplicons of the mtDNA (<250 bp) and compared with two known common deletions (CDs): 4977‐bp and 3895‐bp. It was found that: 1) CD 4977 and CD 3895 were common in all cell lines and in cheek cells. 2) The percentages of the two CDs varied widely, from 0.1 and 3.8 to 2.2 and 14.6% in cell lines and, from 0.1 to 7.8 and 1.6 to 20.4% in cheek cells, for CD 4977 and CD 3895, respectively. Due to large inter‐individual variations, the differences in age groups were not significant (P>0.05). 3) A few deletions (2 ‐8 kb) in cell lines and in cheek cells were demonstrated in gels. In conclusion, we identified and quantified several mtDNA deletions in human cell lines and cheek cells by qPCR. Our results demonstrate that qPCR with optimized conditions could be used as a sensitive, precise, and cost‐effective assay for high throughput screening of mtDNA deletions.