1. Detection of Aberrant TERT Promoter Methylation by Combined Bisulfite Restriction Enzyme Analysis for Cancer Diagnosis 
Seungjae Lee, Sumit Borah, Armita Bahrami 
The Journal of Molecular Diagnostics 
Volume 19, Issue 3, Pages (May 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
COBRA can distinguish hypermethylated from unmethylated UTSS through the PCR-RFLPs generated by the bisulfite treatment of genomic DNA. A: Map showing the positions of 26 CpG dinucleotides located in the proximal TERT promoter [nucleotides to on chromosome 5 (GRCh37/hg19)]. The five CpG dinucleotides within the UTSS, located −485 to −541 bp upstream of the translation start site (ATG), are indicated. The genomic sequence of the UTSS before bisulfite treatment, as well as the resulting sequences after bisulfite conversion if none or all of the CpG dinucleotides in the UTSS are protected by methylation, are shown. CpG dinucleotides are highlighted in red, and BsiWI (5′-CGTACG-3′) and Hpy188I (5′-TCNGA-3′) restriction sites generated by bisulfite treatment are indicated. B: Restriction digestion of the UTSS PCR product from bisulfite-treated unmethylated (lanes 2 to 4) or methylated (lanes 5 to 7) genomic DNA. Dashes above lanes 2 and 5 indicate that no restriction enzyme was added to those reactions. Identical blotches observed on a subset of gels are the result of residue present on the surface on which those gels were imaged.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
A and B: COBRA analysis of UTSS hypermethylation in melanoma samples (A) and proliferative nodules in the giant congenital nevus (B). Dashes indicate that no restriction enzyme was added to those reactions. C: Summary of sequencing results for each of the nine clones analyzed from melanoma case S1001, which was a tumor that was not previously analyzed by high-throughput bisulfite sequencing. Positions of each of the 26 CpG dinucleotides in this region are shown, and the region constituting the UTSS is indicated. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. Asterisks indicate primer dimers approximately 90 bp long. Identical blotches observed on a subset of gels are the result of residue present on the surface on which those gels were imaged.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Detection of TERT promoter methylation patterns in an NBL cohort by COBRA and MassARRAY. A: Representative sequencing results for nine clones obtained from a patient with high-risk (HR) NBL. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively. B: Summary of MassARRAY results for CpG dinucleotide methylation in the TERT promoter. Hypermethylated CpG dinucleotides were identified on the basis of demonstration of ≥10% resistance to bisulfite conversion from cytosine to thymine, and are indicated in blue. CpG dinucleotides for which information was not available are shaded in gray. The tumor risk group was determined by clinical criteria and risk factors. Supplemental Table S1 summarizes the samples that showed evidence of alternative mechanisms of TERT up-regulation or of alternative lengthening of telomeres. C: Restriction patterns of the PCR-amplified product from NBL samples that gave positive signals in COBRA analysis. D: Representative restriction patterns of the PCR-amplified product from samples that gave negative signals in COBRA analysis. Asterisks indicate primer dimers. Dashes indicate that no restriction enzyme was added to those reactions. Identical blotches observed on a subset of gels are the result of residue present on the surface on which those gels were imaged. LR, low-risk.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions