Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues
| Autor: | Chetiya Danthanawanit, Yannawan Wongchai, Puttarakun Meesiri, Sutthirat Udommethaporn, Nunthawut Chat-Uthai, Naravat Poungvarin, Shanop Shuangshoti, Pichpisith Vejvisithsakul, Chinachote Teerapakpinyo |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Male endocrine system diseases Colorectal cancer Thai People Mutagenesis and Gene Deletion Techniques DNA Mutational Analysis Gene Identification and Analysis Oligonucleotides lcsh:Medicine Artificial Gene Amplification and Extension medicine.disease_cause Polymerase Chain Reaction Biochemistry law.invention 0302 clinical medicine law Medicine and Health Sciences Missense mutation Ethnicities lcsh:Science Polymerase chain reaction Mutation Multidisciplinary Nucleotides Melanoma Middle Aged Thailand Oncology 030220 oncology & carcinogenesis Female Colorectal Neoplasms Research Article Proto-Oncogene Proteins B-raf Substitution Mutation Mutation Missense Biology Research and Analysis Methods Proto-Oncogene Proteins p21(ras) 03 medical and health sciences medicine Genetics Humans Molecular Biology Techniques neoplasms Mutation Detection Molecular Biology Aged Colorectal Cancer Point mutation lcsh:R Cancer Biology and Life Sciences Cancers and Neoplasms medicine.disease digestive system diseases 030104 developmental biology Mutational Analysis HEK293 Cells Amino Acid Substitution People and Places Mutation testing Cancer research lcsh:Q Population Groupings |
| Zdroj: | PLoS ONE PLoS ONE, Vol 13, Iss 6, p e0198795 (2018) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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