Highly sensitive EGFR mutation detection by specific amplification of mutant alleles

Leelatian, Nalin; Boonchoo, Pichpisith; Wijitburaphat, Sitsom; Moolsuwan, Kanya; Wongjaroen, Pattara; Chinnasang, Priyakorn; Anyamaneeratch, Komsan; Ruangchira-urai, Ruchira; Poungvarin, Naravat

doi:10.1016/j.yexmp.2013.12.006

Cited by 3 publications

(4 citation statements)

References 29 publications

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“…The final PCR product size is 115 nucleotides. SYBR Green-based allele-specific PCR assays were performed in previously described conditions [ 32 ] with minor modifications. Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction.…”

Section: Methodsmentioning

confidence: 99%

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Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

et al. 2018

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The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

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Section: Methodsmentioning

confidence: 99%

“…We have previously established AS-PCR-based detection of clinically important EGFR mutations in exons 19, 20 and 21 [ 32 ]. In our experience working with SYBR Green I-based AS-PCR, this technique is simple, cost-effective and highly sensitive.…”

Section: Introductionmentioning

confidence: 99%

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

et al. 2018

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“…Very limited biomarkers using IHC and single-gene PCR tests, however, are fully reimbursed to all Thai citizens, including IHC for HER-2 in breast cancer and qPCR for an EGFR mutation in advanced NSCLC. Along with commercially-available genetic testing platforms, Thai researchers have used laboratory-developed tests to reduce the cost and improve the efficiency of the tests 15 , 16 . Examples of commonly used tests to detect genetic alterations in cancer patients available in Thailand are summarized in Table 1 .…”

Section: The Landscape Of Genomic Medicine In Thailandmentioning

confidence: 99%

Genomic medicine and cancer clinical trial in Thailand

Thamlikitkul,

Parinyanitikul,

Sriuranpong

2023

Cancer Biol Med

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“…This difficulty has been addressed by a wide range of molecular techniques for mutation detections. Methods commonly used include restriction enzyme digestion of wild-type DNA [5], [6], [7], peptide nucleic acids (PNA) suppression of wild-type elongation [8], [9], [10], [11], allele-specific amplification [12], [13], [14], sequence-specific ligation [16], and COLD-PCR [17]. More recently, digital PCR based on the compartmentalization and amplification of single DNA molecules [18], [19], [20] and deep sequencing based on next generation sequencing technology [21] are also proposed for increased sensitivity or multiplexity.…”

Section: Introductionmentioning

confidence: 99%

Real-Time Bidirectional Pyrophosphorolysis-Activated Polymerization for Quantitative Detection of Somatic Mutations

Song

Zhong

2014

PLoS ONE

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Detection of somatic mutations for targeted therapy is increasingly used in clinical settings. However, due to the difficulties of detecting rare mutations in excess of wild-type DNA, current methods often lack high sensitivity, require multiple procedural steps, or fail to be quantitative. We developed real-time bidirectional pyrophosphorolysis-activated polymerization (real-time Bi-PAP) that allows quantitative detection of somatic mutations. We applied the method to quantify seven mutations at codons 12 and 13 in KRAS, and 2 mutations (L858R, and T790M) in EGFR in clinical samples. The real-time Bi-PAP could detect 0.01% mutation in the presence of 100 ng template DNA. Of the 34 samples from the colon cancer patients, real-time Bi-PAP detected 14 KRAS mutant samples whereas the traditional real-time allele-specific PCR missed two samples with mutation abundance <1% and DNA sequencing missed nine samples with mutation abundance <10%. The detection results of the two EGFR mutations in 45 non-small cell lung cancer samples further supported the applicability of the real-time Bi-PAP. The real-time Bi-PAP also proved to be more efficient than the real-time allele-specific PCR in the detection of templates prepared from formalin-fixed paraffin-embedded samples. Thus, real-time Bi-PAP can be used for rapid and accurate quantification of somatic mutations. This flexible approach could be widely used for somatic mutation detection in clinical settings.

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Highly sensitive EGFR mutation detection by specific amplification of mutant alleles

Cited by 3 publications

References 29 publications

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

Genomic medicine and cancer clinical trial in Thailand

Real-Time Bidirectional Pyrophosphorolysis-Activated Polymerization for Quantitative Detection of Somatic Mutations

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