title: [Establishment and application of quantitative detection for heteroplasmic mtDNA mutation 3243A→G].
abstract: OBJECTIVE
  To develop a rapid, simple, cost-effective, accurate and sensitive method for quantitative detection of mitochondrial DNA (mtDNA) 3243A→G mutation in order to provide reference for selecting the best detection method under different conditions.
  
  
  METHODS
  Genomic DNA was extracted from peripheral leucocytes of 17 individuals from a Wenzhou family featuring maternally inherited diabetes and deafness (MIDD). Heteroplasmic level of mtDNA 3243A→G mutation was determined respectively with polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP), real time-amplification refractory mutation system-quantitative PCR (RT-ARMS-qPCR) and pyrosquencing. Eleven plasmids with various heteroplasmic levels of the 3243A→G mutation (ranging from 0 to 100%)were constructed as the standards. The reliability of above methods was compared by correlation coefficient based on observed and expected values.
  
  
  RESULTS
  For all three methods, measurement of the standards showed a linear correlation between the expected and detected values, i.e., PCR-RFLP (R(2)=0.828), RT-ARMS-qPCR (R(2)=0.998) and pyrosquencing (R(2)=0.997). For the MIDD family, it was consistent that there are 13 members carrying the A3243G mutation with different heteroplasmic levels. And there was no significant difference between the results by RT-ARMS-qPCR and pyrosquencing.
  
  
  CONCLUSIONS
  PCR-RFLP is not appropriate for the quantitative detection but could be used for early clinical screening. Both RT-ARMS-qPCR and pyrosquencing are suitable for the detection of low heteroplasmic level of A3243G mutation. Compared with pyrosquencing, RT-ARMS-qPCR is rapid, reliable and cost-effective, and is the best choice for detecting low mutation loads.
authors.affiliation: School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, P.R. China. liweiwzmc@163.com.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Zhang
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.forename: Yu
authors.initials: Y
authors.lastname: Yang
authors.forename: Chao
authors.initials: C
authors.lastname: Zheng
authors.forename: Jingbin
authors.initials: J
authors.lastname: Yan
authors.forename: Zhaoning
authors.initials: Z
authors.lastname: Lu
authors.forename: Jianxin
authors.initials: J
authors.lastname: Lyu
authors.forename: Wei
authors.initials: W
authors.lastname: Li
mesh_terms: D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D002648:Child; D004272:DNA, Mitochondrial / Q000235:genetics*; D003638:Deafness / Q000175:diagnosis / Q000235:genetics*; D003920:Diabetes Mellitus / Q000175:diagnosis / Q000235:genetics*; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010375:Pedigree; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D055815:Young Adult
chemical_list: D004272:DNA, Mitochondrial