Mutant Enrichment with 3′-Modified Oligonucleotides

Lee, Seung Tae; Kim, Ji-Youn; Kown, Min-Jung; Kim, Sun Wook; Chung, Jae Hoon; Ahn, Myung‐Ju; Oh, Young Lyun; Kim, Jong-Won; Ki, Chang‐Seok

doi:10.1016/j.jmoldx.2011.07.003

Cited by 36 publications

(16 citation statements)

References 32 publications

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“…LOQ and LOD were defined as the respective lowest DNA concentration that could be quantified or detected with an accuracy of 80% or more in a dilution series of DNA standard fragments, as described in the supplementary material. The nested qPCR incorporates the established second round qPCR as well as a first round PCR using WT outer primers overlapping with the mutation specific inner ARMS primers and a 3′ C6 amine modified WT blocking primer 8 to enrich mutated fragments, reduce background noise, and enhance the concentration of DNA in the template (Supplementary Fig. S1 ).…”

Section: Methodsmentioning

confidence: 99%

“…Different approaches have been presented to detect and quantify ctDNA, such as quantitative real-time PCR (qPCR) using ARMS primers 5 , PNA clamping 6 , LNA primers or probes 7 , blocking primers 8 , HRM analysis 9 , COLD-PCR 10 , and digital PCR methods 4 , 11 , 12 . Furthermore, genome wide or hot spot sequencing approaches covering huge numbers of possible cancer related mutations have been developed 13 .…”

Section: Introductionmentioning

confidence: 99%

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Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection

Ehlert

Tug

Brahmer

et al. 2017

Sci Rep

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The analysis of blood plasma or serum as a non-invasive alternative to tissue biopsies is a much-pursued goal in cancer research. Various methods and approaches have been presented to determine a patient’s tumour status, chances of survival, and response to therapy from serum or plasma samples. We established PNB-qPCR (Pooled, Nested, WT-Blocking qPCR), a highly specific nested qPCR with various modifications to detect and quantify minute amounts of circulating tumour DNA (ctDNA) from very limited blood plasma samples. PNB-qPCR is a nested qPCR technique combining ARMS primers, blocking primers, LNA probes, and pooling of multiple first round products for sensitive quantification of the seven most frequent point mutations in KRAS exon 2. Using this approach, we were able to characterize ctDNA and total cell-free DNA (cfDNA) kinetics by selective amplification of KRAS mutated DNA fragments in the blood plasma over the course of tumour resection and the surrounding days. Whereas total cfDNA concentrations increased over the surgical and regenerative process, ctDNA levels showed a different scheme, rising only directly after tumour resection and about three days after the surgery. For the first time, we present insights into the impact of surgery on the release of ctDNA and total cfDNA.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection

Ehlert

Tug

Brahmer

et al. 2017

Sci Rep

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“…The primer sequences are DIS3 forward: 5′-TTAGCCACTCGCTGTATGATG-3′; DIS3 reverse: 5′-G ATGCACGTTGGGCATATTG-3′; DIS3 sequencing: 5′-GC TTGTGTTTGTCTGTCAACTC-3′; MYO10(5003) forward: 5′-CAGCAAAGGCATCTAACAGAAC-3′; MYO10(5003) reverse: 5′-AGTGAGACATTGGCTCTTTAGG-3′; MYO10 (5003) sequencing: 5′-GTGGGAGTTGATGGTGATCTT-3′; MYO10(5300) forward: 5′-TCACGTAAGACCGTAGCT TTATC-3′; MYO10(5300) reverse: 5′-GATGACCACAC GGGTAACAT-3′; MYO10(5300) sequencing: 5′-ACAG GCATCAACACAGGTAAA-3′. Regions flanking the WM hotspot mutation site MYD88 L265 were amplified and sequenced by MEMO (Mutant Enrichment with 3′-Modified Oligonucleotides) PCR [36] using primers MYD88 blocking: 5′-AAGCGACTGATCCCCATCAA-3′[C3SP]; MYD88 forward: 5′-CAGGTGCCCATCAGAAGC-3′; MYD88 reverse: 5′-GAAGTTGGCATCTCCAGGAA-3′. MYD88 reverse primer was used as a sequencing primer.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive genomic profiling of IgM multiple myeloma identifies IRF4 as a prognostic marker

et al. 2016

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Immunoglobulin M multiple myeloma (IgM MM) is an extremely rare subtype of multiple myeloma with a poor clinical outcome. In this study, bone marrow aspirates of MM patients, including two cases of IgM MM, were analyzed by whole exome sequencing and RNA sequencing. Recurrent somatic mutations in the NRAS, KRAS, CCND1, DIS3, and TP53 genes were found in IgM MM and other types of MM, in agreement with previous studies. Overall transcription profiles of IgM and other types of MM clustered together, but separate from normal blood or peripheral plasma cells. Among the differentially expressed genes in IgM MM, IRF4 was highly expressed in IgM as well as in a subset of other types of MM patients. Thus, IRF4 is an independent prognostic factor for general MM patients. Taken together, the somatic mutation and transcriptome profiles support the idea that IgM MM can be classified as an aggressive MM subtype.

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“…Although conventional Sanger sequencing is the standard method used to detect the BRAF V600E mutation, it is not sensitive enough (approximately 20%) to detect the mutation if it is present at a low frequency in specimens [ 14 ]. Other molecular methods with mutation enrichment that have a higher sensitivity than conventional Sanger sequencing, such as DPO PCR and mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing, have also been developed [ 12 14 15 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the Anyplex BRAF V600E Real-Time Detection Assay Using Dual-Priming Oligonucleotide Technology in Fine-Needle Aspirates of Thyroid Nodules

et al. 2015

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BackgroundSeveral molecular assays have been developed to detect the BRAF V600E mutation in fine needle aspirates (FNAs) for the diagnosis of papillary thyroid cancer. Using a multiplex PCR technique, we evaluated the Anyplex BRAF V600E Real-time Detection (Anyplex) assay and compared its efficacy with that of the Seeplex BRAF V600E ACE Detection (Seeplex) method.MethodsWe tested 258 consecutive FNA specimens using the Seeplex and Anyplex assays. Any conflicting results between the two assays were confirmed by using mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The limits of detection (LODs) and reproducibility for each assay were evaluated with serially diluted DNA from a BRAF V600E-positive cell line.ResultsThe BRAF V600E mutation was detected in 36.4% (94/258) FNA specimens by either the Seeplex or Anyplex assay. Results for the two assays showed 93.4% (241/258) agreement, with a kappa value of 0.861 (95% confidence interval, 0.798-0.923). Of the eight specimens that were BRAF V600E-positive by the Anyplex assay but not by the Seeplex assay, five were found to be BRAF V600E-positive by MEMO sequencing. The mutation detection rate of the Seeplex and Anyplex assays was 79.0% and 84.0%, respectively, in the FNA specimens diagnosed as malignant (n=81). The LOD as determined by probit analysis was 0.046% (95% confidence interval, 0.019-0.532%).ConclusionsThe Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.

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Mutant Enrichment with 3′-Modified Oligonucleotides

Cited by 36 publications

References 32 publications

Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection

Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection

Comprehensive genomic profiling of IgM multiple myeloma identifies IRF4 as a prognostic marker

Evaluation of the Anyplex BRAF V600E Real-Time Detection Assay Using Dual-Priming Oligonucleotide Technology in Fine-Needle Aspirates of Thyroid Nodules

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