Detection Of DNA Mutations Research Articles

DNA Mutation: Ultrasensitive Detection of Mitochondrial DNA Mutation by Graphene Oxide/DNA Hydrogel Electrode (Adv. Funct. Mater. 44/2014)

DNA Mutation: Ultrasensitive Detection of Mitochondrial DNA Mutation by Graphene Oxide/DNA Hydrogel Electrode (Adv. Funct. Mater. 44/2014)

Abstract LB-170: Droplet digital PCR detection and longitudinal monitoring of BRAF mutations in cell-free urinary DNA of patients with metastatic cancers or Erdheim-Chester disease

Abstract Background: Detection and monitoring of oncogenic mutations in cell-free urinary DNA opens the possibility of a new paradigm for a truly non-invasive method of individualized care for metastatic cancer patients, which would enable the quantitation of mutational tumor load and respective concordance to therapeutic responsiveness followed by detection of emerging genomic alterations underlying acquired resistance. Methods: Cell-free DNA was isolated from single and/or multiple sequential urine samples from patients with advanced cancers or Erdheim-Chester disease and BRAF V600E mutation in the tumor tissue from a CLIA-certified laboratory, who progressed on systemic therapy. Assays for quantitative assessment of BRAF V600E mutation in cell-free urinary DNA were developed using digital droplet PCR methodology (RainDance, MA) with enrichment of mutation-containing DNA fragments by pre-amplification of the BRAF gene. Detection limits were established as wild-type (<0.05% of mutant copies), low-mutant (0.05%-0.107%), mutant (>0.107%). Results: Cell-free DNA was extracted from urine of 33 patients with diverse advanced cancers (melanoma, n=11; colorectal cancer, n=8; non-small cell lung cancer, n=4; papillary thyroid carcinoma, n=4; other, n=4) or Erdheim-Chester disease (n=1) with BRAF V600E mutation in the tumor tissue. Of these 33 patients, 25 (76%) had the same mutation in urinary cell-free DNA (mutant, n=14; low-mutant, n=11). In addition, 17 patients had longitudinal analysis of percentage of BRAF V600E mutation to wild-type in sequentially collected urine samples and the dynamics of BRAF V600E mutation in the urine correlated with response to therapy in 13 (76%) of them. Conclusion: Our data suggest that detecting BRAF V600E mutation in cell-free DNA from urine can offer a noninvasive alternative to mutation testing of tumor tissue with acceptable concordance and should be investigated further for testing and monitoring of mutation status in patients with cancer and Erdheim-Chester disease. Citation Format: Filip Janku, Gerald S. Falchook, Sarina A. Piha-Paul, Aung Naing, Veronica R. Holley, David S. Hong, Vivek Subbiah, Daniel D. Karp, Ralph G. Zinner, Siqing Fu, Jennifer J. Wheler, Funda Meric-Bernstam, Vanda M. Stepanek, Rajyalakshmi Luthra, Lorieta Leppin, Latifa Hassaine, Karena Kosco, Jason C. Poole, Cecile Rose T. Vibat, Mark G. Erlander. Droplet digital PCR detection and longitudinal monitoring of BRAF mutations in cell-free urinary DNA of patients with metastatic cancers or Erdheim-Chester disease. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-170. doi:10.1158/1538-7445.AM2014-LB-170

Ultrasensitive Detection of Mitochondrial DNA Mutation by Graphene Oxide/DNA Hydrogel Electrode

Ultrasensitive detection of nucleic acid has attracted considerable attention recently in academic research and clinic diagnostics. Current approaches for DNA analysis involve complicated or expensive processes for labeling and often yield a high detection limit. In this study, a hydrogel electrode prepared from graphene oxide and fish sperm DNA is used for label−free mitochondrial DNA detection by impedimetric approach. The hydrogel has a bionic structure containing rich water and natural biomolecule fish sperm DNA that would benefit the adsorption and hybridization of DNA. Graphene oxide is a semiconductor and its conductivity can be improved by doping negatively charged DNA molecules. The result shows that the conductivity and impedance change of hydrogel electrode could be tuned by its length and component. The linear range for DNA detection by the optimized hydrogel is from 1.0 × 10−9 to 1.0 × 10−20 M with a detection limit of 1.0 × 10−20 M. The result is ascribed to the bionic structure and tunable conductivity of hydrogel electrode. The hydrogel electrode has been used to detect the real DNA samples from patients of ovarian cancer with satisfactory results.

QClamp Liquid Biopsy Technology (QLBT) rapid sensitive detection of somatic mutations in tumor-derived DNA from whole blood.

e22130 Background: In the field of targeted cancer therapy precise knowledge of the genetics of a specific patients tumor is essential for therapeutic decisions. Distant tumor metastases often harbor unique genomic characteristics not detectable in the corresponding primary tumor of the same patient. Thus, the mere analysis of the resected primary tumor alone (current standard practice in oncology) may not reveal sufficient information for treatment decisions. This dilemma can be solved by a new diagnostic concept: liquid biopsy, that is, analysis of therapeutic targets and drug resistance–conferring gene mutations on circulating tumor cells (CTC) and cell-free circulating tumor DNA (ctDNA) released into the peripheral blood from metastatic deposits. QClamp liquid biopsy technology (QLBT) enables CTCs and ctDNA to be effective biomarkers in future oncology trials assessing new drugs or drug combinations. Methods: QClamp is a qPCR method for the direct precise analysis of cancer gene mutations in DNA from formalin-fixed paraffine embedded (FFPE), fresh or frozen tumor biopsy samples or whole blood samples. QClamp utilizes a novel PCR Direct sample preparation method called 'QZol' which only takes 20 min. prior to direct use of an aliquot of the QZol lysate in a real-time PCR reaction. The use of a xeno-nucleic acid (XNA) clamp probe specific for the target wild-type gene sequence allows the genration of only mutation specific amplicons which are detected by the fluorecent interchelating dye SYBR Green. Results: Mutations in the JAK2 gene (V617F) in blood samples from patients suffering from myeloproliferative cancer were detected by QClamp and showed 100% concordance with sequencing data obtained from analysis of the DNA purified from the patients blood samples by a commercial DNA purification kit. Conclusions: QClamp Liquid Biopsy Technology (QLBT) is a direct, rapid, precise and sensitive method for analysis of a cancer patients tumor genetic characteristics form a wide range of clinical samples including FFPE, fresh and/or frozen tumor biopsy samples or from whole blood samples to aid in targeted cancer therapy decisions.

Highlights of This Issue

Id1 induces activation of PI3K/AKT pathway, but the underlying mechanism remains unknown. In this study, Li and colleagues report that Id1 induces the expression and secretion of IGF2 in several cancer types including esophageal cancer. Id1-induced IGF2 could activate the PI3K/AKT pathway by signaling through IGF1R in an autocrine and endocrine manner to promote tumor progression and metastasis. The study also shows that IGF2 and IGF1R are valid targets in esophageal cancer therapy and that a monoclonal antibody targeting IGF1R (cixutumumab), used alone or combined with cytotoxic chemotherapeutic drugs, has potential clinical applications in systemic treatment of esophageal cancer.Beaver and colleagues demonstrate the ability to detect DNA mutations in plasma from early-stage breast cancer patients. In a prospective study, blood from 29 patients was obtained prior to surgery and used to detect PIK3CA mutations with 93.3% sensitivity and 100% specificity. Five patients had plasma PIK3CA mutations after surgery. This study demonstrates the feasibility of detecting plasma tumor DNA in early-stage breast cancer. Importantly, this provides the foundation for future work examining whether persistent plasma tumor DNA postsurgery could identify patients who may benefit from adjuvant therapies versus those that are already cured after surgery.Follicular lymphomas (FL) establish an inflammatory microenvironment, notably driven by COX-2, with a large amount of PGE2 synthesis. To inhibit the crosstalk between the supporting tumor microenvironment and tumor B-cells, Gallouet and colleagues showed that the use of celecoxib, an anti-inflammatory drug, can reduce in vitro PGE2 production from FL-patients stromal cells. Moreover, the use of delecoxib and TRAIL, a cytotoxic molecule, can sensitize primary B-cells from FL patients to death. This combination of treatments can therefore target both microenvironment and tumor cells for more efficiency in FL treatments.Although detection of low levels of human cytomegalovirus (CMV) expression in glioblastoma (GBM) has been reported by several groups, the physiologic relevance of this expression remains unclear. Nair and colleagues explored whether the CMV antigen pp65 could serve as an effective tumor rejection antigen in primary GBM. Despite deficits in cellular immunity in patients with GBM, CMV pp65-specific T cells could be reliably activated in vitro using pp65 RNA-pulsed autologous dendritic cells. Importantly, CMV-specific T cells were effective in recognizing and killing autologous GBM tumor cells. These studies strongly support the rationale for CMV-targeted immunotherapy in patients with GBM.

Automated Objective Determination of Percentage of Malignant Nuclei for Mutation Testing

Detection of DNA mutations in tumor tissue can be a critical companion diagnostic test before prescription of a targeted therapy. Each method for detection of these mutations is associated with an analytic sensitivity that is a function of the percentage of tumor cells present in the specimen. Currently, tumor cell percentage is visually estimated resulting in an ordinal and highly variant result for a biologically continuous variable. We proposed that this aspect of DNA mutation testing could be standardized by developing a computer algorithm capable of accurately determining the percentage of malignant nuclei in an image of a hematoxylin and eosin-stained tissue. Using inForm software, we developed an algorithm, to calculate the percentage of malignant cells in histologic specimens of colon adenocarcinoma. A criterion standard was established by manually counting malignant and benign nuclei. Three pathologists also estimated the percentage of malignant nuclei in each image. Algorithm #9 had a median deviation from the criterion standard of 5.4% on the training set and 6.2% on the validation set. Compared with pathologist estimation, Algorithm #9 showed a similar ability to determine percentage of malignant nuclei. This method represents a potential future tool to assist in determining the percent of malignant nuclei present in a tissue section. Further validation of this algorithm or an improved algorithm may have value to more accurately assess percentage of malignant cells for companion diagnostic mutation testing.

Detection of DNA mutations by PCR-TTGE method

In our study PCR-temporal temperature gelelectrophoresis (TTGE) and MeltINGENY bioinformatic program were used to analyse the mutations in the genes of melanocortin-1 receptor (MC1R) and pituitary adenylate-cyclase activating polypeptide (PACAP) in cattle. Amplification of target DNA by PCR was performed with GC-clamp primers and non-GC-clamp primers in simplex PCR reactions. The fragments were separated by denaturing polyacrylamide gelelectrophoresis (denaturing agents: high temperature, urea) after PCR reactions.MC1R homozygous individuals were used for the reaction.
 We concluded that MeltINGENY program makes the decision and detection system easier, and more simple as the melting profile of target sequence is determined by the software. In case of MC1R gene, PCR-TTGE method is appropriate for SNP detection, however PACAP gene polymorphism can not be identified by the method, because PACAP mutations are not included in melting domains, therefore PCR-TTGE cannot detect them.

Rapid detection of DNA by magnetophoretic assay

Recently, marine environmental pollution has become more and more serious due to oil spills or the collapse of nuclear power plant resulting in a massive release of hazardous materials to the ocean. These harmful radioactive materials are associated with the random mutation of genes leading to deformity of individuals. So far there has been no effective system for monitoring this marine pollution. Therefore, rapid detection of DNA mutations has become increasingly important in a variety of fields including medical diagnostics and food safety. Herein, we report a rapid, sensitive, qualitative and quantitative approach for DNA detection based on the magnetophoretic assay. The Au-magnetic microparticles (MMPs) nanocomplex was used to generate a color signal and uncombined Au NPs contributed an optical signal that could be directly quantified by UV–Vis absorption spectroscopy of the supernatant after the sample was placed in a magnetic field for 2min. The linear relationship between signal intensity and the target DNA concentrations (0.1nM–1.0μM) was investigated and a limit of quantitation of 0.1nM was achieved using this method.

Introducing the 2014 JALA Ten Honorees

Introducing the 2014 JALA Ten Honorees

Detection of Mitochondrial Mutations in Cancer by Next Generation Sequencing

Mitochondria play a central role in the regulation of cellular function, metabolism, and cell death in many types of cell including cancer. Little attention has been paid to the potential mutations that can affect mitochondrial function in cancer, other than specific mutations in genes that are encoded in the nuclear DNA and so once mutated, all mitochondria in a cell will be affected. However, both somatic and germline mutations in mitochondrial genes are common and have been suggested to contribute to the development of cancer. Recent advances in high-throughput technologies have allowed for the rapid and accurate detection of mitochondrial DNA (mtDNA) mutations, polymorphisms, or copy number variations in a variety of tissues and bodily fluids, and can be used as a tool for early detection and treatment of cancer. In addition to deep-sequencing specifically targeted at mtDNA, mtDNA sequences can also be extracted from exome (or whole genome) sequencing data.

Enhanced Sensitivity for Detection of Low-Level Germline MosaicRB1Mutations in Sporadic Retinoblastoma Cases Using Deep Semiconductor Sequencing

Sporadic retinoblastoma (RB) is caused by de novo mutations in the RB1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by Sanger sequencing. Next-generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA. Deep sequencing of the RB1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where Sanger sequencing excluded the presence of mutations. The individual exons of the RB1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an Ion Torrent Personal Genome Machine. Six low-level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low-level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB. The use of deep sequencing technology increased the sensitivity of the detection of low-level germline mosaic mutations in the RB1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB.

Digital PCR as a Novel Technology and Its Potential Implications for Molecular Diagnostics

The latest incarnation of PCR, digital PCR (dPCR),2 takes 2 decades of development in enzyme chemistry and assay design and applies them with formidable precision and sensitivity. dPCR is achieved by performing a limiting dilution of DNA into a succession of individual PCR reactions (or partitions). Limiting dilution, made practical by advances in partitioning with nanofluidics and emulsion chemistries, capitalizes on the random distribution of the DNA template and the fact that Poisson statistics can be used to measure the quantities of DNA present for a given proportion of positive partitions. And what is more, it appears to work; results obtained with the technique are linear, and it is capable of detecting and quantifying miniscule amounts of template (1, 2). All of these features are achievable without the calibration curve required with almost all other molecular methods for accurately quantifying DNA. Compared with real-time quantitative PCR (qPCR), dPCR has already been heralded as more precise (3), better at detecting rare genetic variants (4), and less susceptible to inhibitors (5, 6). Recognition of these advantages has naturally led to speculation as to the potential of dPCR for molecular diagnostics. This issue of Clinical Chemistry presents the reports of 2 studies that have demonstrated the unique clinical application of dPCR for measuring circulating cell-free nucleic acids. Taly et al. (7) build on their group's leading research in the application of dPCR to investigating the detection of rare tumor-associated mutations in cell-free DNA (cfDNA) in the plasma of cancer patients, and Beck et al. (8) report that the cfDNA of transplantation patients contains detectable quantities of DNA from donor organs and that monitoring of such DNA may serve as a surrogate marker of graft injury and rejection. The articles demonstrate the clinical application of 2 aspects of dPCR, namely …

Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours

Massively parallel sequencing offers the ability to interrogate a tumour biopsy for multiple mutational changes. For clinical samples, methodologies must enable maximal extraction of available sequence information from formalin-fixed and paraffin-embedded (FFPE) material. We assessed the use of targeted capture for mutation detection in FFPE DNA. The capture probes targeted the coding region of all known kinase genes and selected oncogenes and tumour suppressor genes. Seven melanoma cell lines and matching FFPE xenograft DNAs were sequenced. An informatics pipeline was developed to identify variants and contaminating mouse reads. Concordance of 100% was observed between unfixed and formalin-fixed for reported COSMIC variants including BRAF V600E. mutations in genes not conventionally screened including ERBB4, ATM, STK11 and CDKN2A were readily detected. All regions were adequately covered with independent reads regardless of GC content. This study indicates that hybridisation capture is a robust approach for massively parallel sequencing of FFPE samples.

18 F-fluorodeoxyglucose-PET as a Biomarker in Huntington's Disease

assessed in a previous paper, progressive neuropathology is responsible for brain tissue loss and functional decline, which is faster in the early stages of disease [5]. Since the age of disease onset may start both at the age of approximately 30–40 years and at very young age, predictive genetic tests can be used to identify subjects genetically predisposed to the disease, but still in the preclinical stage. Despite the complexity of the procedures, genetic testing is now available throughout the world and governed by international guidelines [6]. When effective treatments are made available, detection of DNA mutations will allow identif ication of subjects who could benefit from neuroprotective therapies. Imaging techniques have greatly contributed to the understanding of the pathophysiology of Huntington’s disease, confirming findings from previously brain postmortem studies of patients after death. Structural MRI and molecular imaging, as performed by F-fluorodeoxyglucosePET has been developed as a valuable biomarker that can be used as an additional

Detection of mitochondrial DNA mutations by high-throughput sequencing in the blood of breast cancer patients

Mitochondrial DNA mutations have been identified in serveral types of cancer. In breast cancer, germline and somatic mitochondrial DNA (mtDNA) mutations have been identified. A number of mtDNA mutations in breast cancer have been identified in protein-coding regions (in protein-coding genes, such as ND2, COX3, ND4, ND5 and CytB). Mutations in these structure proteins cause impaired electron transport function and lead to electron leakage and increased reactive oxygen species (ROS) production, which in turn increases oxidative stress and oxidative damage to the mitochondria, as well as to cells. These data establish an association between mtDNA mutations and breast cancer; however, there is no reliable prediction of breast cancer predisposition or progression based on mtDNA mutation patterns thus far. In this study, we used high-throughput sequencing to detect mtDNA mutations in the blood of breast cancer patients. Some of these mutations may be used as potential markers for breast cancer diagnosis.

Abstract B175: Detection and monitoring of BRAF and KRAS mutations in cell-free urinary DNA of metastatic cancer patients by droplet digital PCR.

Abstract Background: Detection and monitoring of oncogenic mutations in cell-free urinary DNA opens the possibility of a new paradigm for a truly non-invasive method of individualized care for metastatic cancer patients, enabling the quantitation of mutational tumor load and respective concordance to therapeutic responsiveness followed by detection of emerging genomic alterations underlying acquired resistance. Methods: Cell-free DNA was isolated from single and/or multiple sequential urine samples from patients with advanced cancers and BRAF V600E, KRAS G12D or G12V mutations in the tumor tissue from a CLIA-certified laboratory, who progressed on systemic therapy. Assays for quantitative assessment of BRAF V600E, KRAS G12D and G12V mutations in cell-free urinary DNA were developed using droplet digital PCR methodology (RainDance, MA) with enrichment of mutation-containing DNA fragments by pre-amplification of BRAF and KRAS genes. Mutation sensitivity of at least 0.03% was achieved by spike-in experiments of input DNA from cell-lines containing BRAF and KRAS mutations. Healthy controls (N=6) yielded baseline signals that were ∼10-fold less than observed for 0.03% sensitivity. Results: Cell-free DNA was extracted from urine of 25 patients with diverse advanced cancers (colorectal cancer, n=8; melanoma, n=7; non-small cell lung cancer, n=6; papillary thyroid carcinoma, n=2; appendiceal carcinoma, n=1; and glioblastoma, n=1) with BRAF V600E (N=18), KRAS G12D (N=5) and KRAS G12V (N=2) in the tumor tissue. Of 18 patients with BRAF V600E mutations in the tumor, 17 (94%) had the same mutation in urinary cell-free DNA. In addition, all 5 (100%) patients with KRAS mutations (G12D, n=5; G12V, n=2) in the tumor tissue DNA had these same mutations in urinary cell-free DNA.A total of 5 patients with BRAF V600E mutations had longitudinal analysis of percentage of cell-free urinary DNA BRAF V600E mutation to wild-type in sequentially collected urine samples. Although the numbers are small the detected amount of BRAF mutant copies are in agreement with a clinical course. Conclusion: Our preliminary data suggest that detecting BRAF V600E, KRAS G12D, and G12V mutations in cell-free DNA from urine can offer a noninvasive alternative to mutation testing of tumor tissue with excellent concordance, and should be investigated further for testing and monitoring of mutation status in patients with cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B175. Citation Format: Filip Janku, Gerald S. Falchook, Sarina A. Piha-Paul, Aung Naing, Apostolia M. Tsimberidou, Veronica R. Holley, Daniel D. Karp, Ralph G. Zinner, Siqing Fu, Jennifer J. Wheler, David S. Hong, Funda Meric-Bernstam, Vanda M. Stepanek, Rayjalakshmi Luthra, Lorieta Leppin, Latifa Hassaine, Karena Kosco, Jason C. Poole, Mark G. Erlander. Detection and monitoring of BRAF and KRAS mutations in cell-free urinary DNA of metastatic cancer patients by droplet digital PCR. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B175.

Abstract B177: Non-invasive tumor mutation detection of cell-free urinary DNA using massively parallel deep DNA sequencing.

Abstract Background: Previous studies have demonstrated the ability to detect tumor DNA mutations by PCR methodologies within cell-free urinary DNA in metastatic cancer patients. This has opened up the possibility to use massively parallel deep sequencing approaches for more global profiling of tumor mutations using cell free urinary DNA from these patients. Methods: In order to achieve clinical utility the sequencing of cell-free urinary DNA must overcome two technical hurdles: 1) the need for extremely high sensitivity, between 0.01-0.05% and 2) an ultra-short DNA footprint (∼30 bp). Here we report the development of a method using cell free DNA extracted from urine that enriches for extremely low levels of mutated tumor DNA and thereby provides a high detection sensitivity. Detection of the KRAS gene mutations were used as a model system; the developed assay utilizes a 31bp footprint, contains a pre-amplification step that specifically enriches mutated DNA fragments and detects 8 KRAS mutations at the codon 12 and 13 sites. Spiking experiments with DNA derived from cell lines harboring the KRAS G12D mutation were completed to determine the limits of sensitivity of our method. Results: Mutant DNA input at amounts of 0.2%, 0.05%, 0.01% and 0.0% of the total DNA returned observed detection levels of 18.25%, 4.45%, 1.84% and 0.54% respectively as a percentage of the total sequence reads with the detection of mutant DNA of 0.01% within the theoretical limit of the assay. To test our assay, we sequenced 30 ng of extracted urinary DNA from a stage IV colorectal carcinoma patient with a known mutation at the G12D site. Enrichment of the mutation presented at a level of 13.06% of our total sequence, corresponding to an input amount of approximately 0.14% mutational load in the patient's urine. Ongoing analysis of a larger number of patients will be presented to further evaluate the clinical sensitivity and specificity of KRAS mutations. Conclusion: Further development of massively parallel DNA sequencing to detect mutations from cell-free urinary DNA has the potential to monitor metastatic patients for response, non-response and the emergence of resistance mechanisms to molecularly targeted therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B177. Citation Format: Jason C. Poole, Filip Janku, Veronica R. Holley, Jennifer J. Wheler, Funda Meric-Bernstam, Rayjalakshmi Luthra, Lorieta Leppin, Lafifa Hassaine, Karena Kosco, Mark G. Erlander. Non-invasive tumor mutation detection of cell-free urinary DNA using massively parallel deep DNA sequencing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B177.

Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis

The factors maintaining genomic integrity, which have been studied in detail in other species, have yet to be investigated in plants. Recent progress in gene-silencing technology has made it possible to produce transgenic plants with loss-of-function phenotypes for the effective analysis of these factors, even with the high redundancy of genes in plants. Therefore, a mutation-detection system for plants is necessary to estimate the biological function of a target gene for mutation frequencies and spectra. Here, we reported the development of a novel system to analyze mutations in the chromosomal DNA of plants. The supF gene of E. coli was used as a target for the mutation because it was possible to detect all mutational base changes. Based on the plasmid pTN30, which carries supF, we constructed a binary Ti vector for its introduction to Arabidopsis genomes. The system was validated by measuring mutations in both non-treated and mutagen-treated transgenic plants. DNA fragments including pTN30 were rescued from the plants, and introduced into E. coli KS40/pOF105 to isolate the supF mutant clones conferring both nalidixic acid and streptomycin resistance on transformants. We found that the mutation frequency was approximately three times higher with the ethyl methanesulfonate (EMS) treatment than without it and G:C to A:T transitions dominated, which was the most reasonable mutation induced by EMS. These results show that this system allowed for the rapid analysis of mutations in plants, and may be useful for analyzing plant genes related to the functions of genomic stability and monitoring environmental genotoxic substances.

Multiplex Detection of DNA Mutations by the Fluorescence Fingerprint Spectrum Technique

A fingerprint spectrum technique that utilizes cationic conjugated-polymer-based fluorescence resonance energy transfer (FRET) is used for multiplex detection of DNA mutations. This method detects as low as 5 % mutation of the total DNA. Ten PIK3CA mutations originating from 30 clinical breast cancer samples could be detected.

Multiplex Detection of DNA Mutations by the Fluorescence Fingerprint Spectrum Technique

Ein spektraler Fingerabdruck für die parallele Erkennung von DNA-Mutationen wird mithilfe des resonanten Fluoreszenzenergietransfers (FRET) unter Verwendung eines kationischen konjugierten Polymers erstellt. Die Methode weist bis hinab zu 5 % Mutation bezogen auf die gesamte DNA nach. Zehn PIK3CA-Mutationen von 30 klinischen Brustkrebsproben wurden erkannt.