1. Pyrosequencing Is an Accurate and Reliable Method for the Analysis of Heteroplasmy of the A3243G Mutation in Patients with Mitochondrial Diabetes 
Jing-bin Yan, Rong Zhang, Can Xiong, Cheng Hu, Yao Lv, Cong-rong Wang, Wei-ping Jia, Fanyi Zeng 
The Journal of Molecular Diagnostics 
Volume 16, Issue 4, Pages (July 2014)
DOI: /j.jmoldx
Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Identification of heteroplasmy in artificial standard samples with Sanger sequencing, PCR-RFLP, and HRM. A: Eight standard samples with different proportions of the A3243G mutation (0%, 2%, 5%, 10%, 50%, 80%, 90%, and 100%) were detected by Sanger sequencing. The mutant site at position 3243 of mtDNA is shown by arrows. B: The same eight standard samples as described in A were also analyzed by PCR-RFLP. The length of the PCR amplicon was 206 bp. A restriction enzyme site (ApaI) that was present only in the mutant samples led to the production of two short fragments (112 and 94 bp) after restriction enzyme digestion. C: Eleven standard samples (0%, 2%, 5%, 10%, 20%, 30%, 50%, 70%, 90%, 95%, and 100%) were analyzed by HRM. The wild-type sample (0%) was chosen to set the baseline, and the differentiated melting curves are shown.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Optimization of the pyrosequencing protocol by changing the input sequence. Artificial standard samples were used to optimize the protocol of pyrosequencing by changing the input sequence. A: We set the input sequence according to the conventional mode; that is, AG was inputted at the mutation position (shading). B: Low heteroplasmy could be distinguished when the input sequences were changed to GAG at the mutation position. Unfortunately, large errors appeared between the MV and AV for samples with high heteroplasmy. C: When the input sequences were changed to GGGAG, the errors between MV and AV were substantially decreased for all of the standard samples (3% versus 2%; 13% versus 10%; 49% versus 50%; 74% versus 80%; 95% versus 100%).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Construction of a cubic polynomial curve that was based on pyrosequencing analysis of artificial heteroplasmy standard samples. To determine the error between the MV and the AV in the pyrosequencing experiment with an input of GGGAG, 14 standard samples with different proportions (0%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%) were analyzed in quadruplicate. A: Representative analyses show slight differences between the MV and AV for all of the standard samples. The results from eight standard samples were shown. The shaded areas represent the region in which the heteroplasmy of A3243G mutation would be calculated. B: The results from the analysis described in A were used to generate a cubic polynomial curve; the equation for this curve is shown. The dots represent the individual replicates.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Identification of heteroplasmy for the A3243G mutation in leukocyte DNA from the pedigree 1096 with both HRM and pyrosequencing. A: Ten peripheral blood samples were collected from a four-generation pedigree with mitochondrial diabetes. The clinical features were obvious in four patients (1096-1, , , and ). Two spouses ( and ) did not have mitochondrial diabetes, but they had the clinical phenotype of type 2 diabetes. B: The heteroplasmy of leukocyte DNA was identified by HRM on the basis of comparisons of the melting curves generated from the artificial heteroplasmy standard samples and those generated from the individual DNA samples from the pedigree. The level of heteroplasmy was approximately 20%, 40%, and 40% in , , and , respectively. The level of heteroplasmy of , , , and was low; we could not confirm the values in these samples by HRM. C: Heteroplasmy was also detected by pyrosequencing, and the values were adjusted on the basis of the cubic polynomial curve equation.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions