1. Mutant Enrichment with 3′-Modified Oligonucleotides
Seung-Tae Lee, Ji-Youn Kim, Min-Jung Kown, Sun Wook Kim, Jae Hoon Chung, Myung-Ju Ahn, Young Lyun Oh, Jong-Won Kim, Chang-Seok Ki 
The Journal of Molecular Diagnostics 
Volume 13, Issue 6, Pages (November 2011)
DOI: /j.jmoldx
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
The concept of MEMO-PCR and an example of the resulting enrichment. A: The 3′-end of a blocking primer, which perfectly matches the wild-type sequence and encompasses the target mutation site, is modified with extension-inhibiting compounds such as a C3 spacer, a C6 amine, or a phosphate. The overlapping generic primer (forward primer in the figure) neighbors the mutation site and is in competition with the blocking primer for DNA binding. For normal sequences, the DNA binding of the blocking primer, which has a higher Tm and a higher concentration, dominates that of the generic primer. B: For mutant sequences, the mismatches between the blocking primer and the target sequence result in reduced affinity and increase the chance for annealing of overlapping generic primers, enabling the enrichment of mutant sequences. C: Sanger sequencing can be performed using the nonoverlapping (reverse) primer, and the example of TP53 R273C (c.817C>T) shows a typical pattern; the absence of wild-type peaks (C peak indicated with arrow) in nondiluted or modestly diluted specimens (1 × 100 and 1 × 10−2 in the figure) due to the near-complete blockage of wild-type sequences and the presence of heterozygous peaks in more highly diluted specimens (1 × 10−4).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Primers and experimental conditions correlate with sensitivity. A: Sensitivity was improved when the amount of blocking primers increased in excess of the amount of generic primers, reaching a plateau around the ratio of 5:1 (50 pmol of blocking primers per 10 pmol of generic primers). B: The ΔTm correlated well with the sensitivity for detecting different mutations on the KRAS codon 12. C: For small deletion/insertion mutations, blocking primers with a higher Tm generally yielded superb sensitivities.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Potential applicability of MEMO to quantitative real-time PCR and/or subsequent melting curve analysis. A: An example of serially diluted specimens with EGFR T790M mutations detected through a real-time PCR assay using a DNA-intercalating fluorescence dye illustrates different fluorescence curves according to the mutant allele concentration. B: The standard curves generated in quadruplicate runs have a very linear correlation (r2 = 0.991) in dilutions ranging from 1.0 × 100 to 1.0 × 10−3 (PCR efficiency: 1.45). C: Subsequent high-resolution melting curve analysis demonstrates a higher Tm (84.3°C to 84.4°C) in dilutions with a high mutant allele concentration (1.0 × 100, 1.0 × 10−1, and 1.0 × 10−2) compared to that of normal samples (83.7°C), whereas samples with low mutant allele concentrations (1.0 × 10−3) produce heterozygous melting curves. D: Amplicons were sequenced, and the results were in concordance with those of the high-resolution melting curve analysis (ie, homozygous mutant peaks in samples with a high mutant allele concentration and heterozygous peaks in those with a low mutant allele concentration). The target mutation site is indicated with an arrow.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Mutations identified by MEMO-PCR. A and B: MEMO-PCR with fluorescence primers and subsequent fragment analysis identifies a 15-bp deletion in EGFR exon 19 at a 1.0 × 10−6 dilution and a 4-bp insertion in NPM1 exon 12 at a 1.0 × 10−5 dilution. MEMO-PCR and pyrosequencing demonstrates increased sensitivities in the specimens with KRAS mutations. C: KRAS c.34G>A (G12S) in 1.0 × 10−2. D: KRAS c.35G>T (p.G12V) in 5.0 × 10−3. E: KRAS c.34G>T (p.G12C) in 5.0 × 10−3. F: KRAS c.35G>C (p.G12A) in 5.0 × 10−2. G: KRAS c.35G>A (p.G12D) in 5.0 × 10−2. H: KRAS c.38G>A (G13D) in 2.0 × 10−2 of allele minorities. Arrows indicate mutant sequences enriched.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions