Mutation identification DNA analysis system (MIDAS) for detection of known mutations

For the detection of point mutations (or variant alleles), PCR followed by restriction fragment length polymorphism (RFLP) detection is widely used and is probably one of the simplest methods for detection of known mutations. This method depends on the fact that the mutation of interest disrupts a recognition sequence for a restriction enzyme. When a restriction site is not present around the mutation, mismatch primers need to be designed that do create a restriction site. Such a mismatch primer is thus usually located directly adjacent to the mutated nucleotide. We validate our PCR-RFLP assays by direct sequencing of the PCR product. In two of our assays, however, we found that none of the three samples found to be heterozygous by PCR-RFLP was confirmed as heterozygous by direct sequencing. Detection of the CYP3A53 polymorphism (A6986G) is based on amplification of a 293-bp DNA fragment digested with Ssp 1 (1). For amplification, we used 40 pmol of primers P1 (5′-CATGACTTAGTAGACAGATGAC-3′) and P2 (5′-GGTCCAAACAGGGAAGAAATA-3′) in a final volume of 50 μL. The reverse primer P2 [which contains a mismatch (underlined nucleotide) to create a restriction site for the Ssp 1 restriction enzyme] is located directly next to the mutated nucleotide. Fig. 1A⇓ shows the …

With the assistance of improved base stacking hybridization, a low-density microarray, containing 12 capture probes, was used to identify 7 known hypertrophic cardiomyopathy-related mutations in the gene of MYH7 (beta-myosia heavy chain). The hybridization targets, amplified from 11 plasmids containing wild type or mutation sequences of MYH7 and healthy genomic DNA, were prepared by single-step fluorescence labeled asymmetric PCR. Six single base substitutions and a trinucleotide deletion were identified unambiguously, and the average discrimination ratio (Qmut) for artificial heterozygous samples was as high as 16.2.

Proof-reading PCR (PR-PCR) is designed to detect known mutations within genomic DNA. It differs from standard PCR approaches in that one of the two primers has its 3' end aligned with a putative mutation site, and has its 3'-OH replaced by a blocking group. Distinguishing a mutant gene from wild-type depends upon preferential removal of the blocked 3' terminal nucleotide by the polymerase proof-reading activity when it is mismatched with the template. Preferential removal of the blocked nucleotide allows subsequent extension and selective amplification, and provides the basis for distinguishing mutant from normal genes. This method has been used here to detect a transition mutation within the P53 gene of HaCaT cells with verification by direct sequencing of the selectively amplified DNA.

Allele specific PCR with microfluorometry: application to the detection of del F508 mutation in cystic fibrosis

Time Course of Acquisition of a CSF3R Mutation and Subsequent Development of AML in a Patient with Cyclic Neutropenia

Mutational Concordance from Diagnosis and Relapse in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group

BACKGROUND: β-thalassemias are widely distributed in Mediterranean and Middle Eastern countries, including Iraq. There are more than 400 transfusion-dependent β-thalassemia patients registered in the thalassemia center. β-thalassemia is a significant problem in Karbala as well as other regions of Iraq. The detection of the most frequent mutations is significant to the implementation of an effective preventive program in this area because of the significant burden it places on the local health authorities, patients, and their families. OBJECTIVES: To define the most common mutations and their frequencies among patients with transfusion-dependent β-thalassemia and to evaluate the reverse hybridization strip assay method for the detection of β-thalassemia mutations. PATIENTS, MATERIALS AND METHODS: Sixty transfusion-dependent β-thalassemia patients were recruited from the thalassemia center in Karbala. Blood samples were aspirated from each patient just before blood transfusions for CBC, reticulocyte count, DNA extraction, PCR amplification, and identification of the mutations by reverse hybridization technique using the β-Globin strip assay method. RESULTS: A total of 60 patients with 120 chromosomes were studied, searching for the most common mutations causing β-thalassemia. Among the twelve identified mutations, the six most frequent mutations represented 79.16% of all β-globin defects. These mutations were IVSII-1 (30.83%), IVSI-110 (15.83%), Codon 5 (10.83%), Codon 44 (8.33%), IVSI-1 (6.67%), and IVSI-5 (6.67%). The detection rate of the method used in our population was 96.66%. CONCLUSION: The most frequent mutations encountered were IVSII.1 and IVSI-110, while IVS 2.745 was the least common mutant allele. Reverse hybridization strip assay molecular techniques used in the current study provide an extremely quick, precise, and simple to carry out molecular diagnostic technique for the detection of β-thalassemia mutations.

Detection of proximal colorectal cancers through analysis of faecal DNA

Digital PCR detection of EGFR somatic mutations in non-small-cell lung cancer formalin fixed paraffin embedded samples

Chapter 4 - Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis

Genetic testing has been available for Huntington's disease for longer than any other adult-onset genetic disorder. The discovery of the genetic mutation causing Huntington's disease allowed the use of predictive testing to identify currently unaffected carriers. Concerns have been raised that predictive testing may lead to an increase in deaths by suicide among identified carriers, and these concerns prompted research to assess the psychological effect of predictive testing for Huntington's disease. This article provides an overview of the literature and draws implications for clinical practice. About 10% to 20% of people at risk for the disease request testing when approached by registries or testing centers. Most available evidence suggests that noncarriers and carriers differ significantly in terms of short-term, but not long-term, general psychological distress. Adjustment to results depended more on psychological adjustment before testing than the testing result itself. Although risk factors for psychological sequelae have been identified, few adverse events have been described, and no obvious contraindications for testing people at risk have been identified. The psychological effects of testing may depend on whether testing was based on linkage analysis or mutation detection. Cohorts enrolled in mutation detection programs have higher levels of depression before and after testing than people who sought genetic testing when linkage analysis was available. Some evidence shows that people who choose to be tested are psychologically selected for a favorable response to testing. The effect of testing on people in settings where less intensive counseling protocols and eligibility criteria are used is unknown. Therefore, genetic testing is best offered as part of comprehensive specialist counseling. The frequency of Huntington's disease in populations of European descent is between 3 and 7 affected people per 100,000 population.1 The mean age of onset of Huntington's disease is 40 years, with a mean duration of 15 years. About 98% of patients with Huntington's disease have an expansion of a trinucleotide (CAG) repeat on the Huntington gene. No medical treatments to delay the onset of disease or prophylactic strategies are available. The discovery of a genetic marker for Huntington's disease in 1983 allowed the use of linkage analysis to identify currently unaffected carriers with a sensitivity of about 96% to 99%.2 Linkage analysis takes advantage of the spatial proximity of a marker and a mutation and requires blood specimens from both affected and unaffected family members from several generations. The nature of linkage analysis is probabilistic, because genetic recombination can occur between markers and the gene during meiosis. Most carriers and noncarriers were given risk estimates of 90% or more and 10% or less, respectively, reflecting the relatively low accuracy of these earlier linkage studies. Since identification of the responsible gene mutation in 1993,3 direct mutation detection methods have been applied, leading to highly accurate predictive testing with a sensitivity and specificity of virtually 100%. Compared with other dominantly inherited genetic traits leading to adult-onset disorders, predictive testing for Huntington's disease has been offered for the longest time.4 Carriers will develop the disease—that is, Huntington's disease confers 100% penetrance, which refers to the proportion of carriers of a genetic alteration who will manifest the effects of it and is equivalent to lifetime incidence. Noncarriers definitely will not develop the disease. When linkage analysis first became available, concerns were raised over the possibility of predictive testing leading to an increase in deaths by suicide among identified carriers. Suicide is well recognized as a serious consequence of Huntington's disease. Results from a carefully designed study showed a 4-fold increase in the suicide rate among people affected compared with the rate of the general white population in the United States.5 Attitudinal surveys of people at risk suggest that between 11% and 33% of participants considered suicide a possible response in the future.6 These data highlighted the need for research to identify predictors of depression and suicidal intention in people entering predictive testing programs for Huntington's disease. The identification of the genetic marker prompted investigation of attitudes to, and the psychological impact of, testing for the disease.

Background The deregulation of the PI3K/AKT signaling pathway is essential to malignant cellular processes of breast cancer, including proliferation, apoptosis, and drug response. Oncogenic activating somatic mutations in the PI3K/AKT pathway are pervasive. However, it remains difficult to discriminate between driver and passenger mutations. This study was conducted to identify the landscape of genetic mutations in the PI3K/AKT pathway using Amplicon Sequencing in a Chinese population. Notably, we developed a Gateway-based mutation barcoding (GaMB) library which enables a high-throughput mutation-phenotype screen for specific vulnerable mutations that contribute to the cancer development and drug resistance. Method We collected 149 breast cancer specimens in a Chinese population and performed Ion Torrent Amplicon Sequencing for the key genes in PI3K/AKT pathway: PIK3CA, PIK3R1, AKT1, AKT2, AKT3, PTEN, PDK1, and the canonical tumor suppressor gene TP53, at 1000× coverage. Next, we established a high-throughput GaMB library that contained all of the PIK3CA mutations, either newly identified in Chinese population or reported in TCGA and COSMIC database, and tagged each mutations with a specific barcode. We then applied this library to functional screening processes using proliferation and drug response selection (doxorubicin or BKM-120) assays through which we screened the functional mutations with specific characteristics. The genomic DNA of the pooled surviving cells from the library, as well as the original cells before the screenings, was extracted and used for PCR amplification of the barcode regions, and then detected using Illumina Miseq sequencing to analyze the functional mutations. We then validated the cellular 2D- & 3D- proliferation abilities and the status of PI3K/AKT pathway activation in presence of identified mutations, respectively. Result Mutations in the PIK3CA (44%), PIK3R1 (37%), AKT3 (15%) and PTEN (12%) genes were the most prevalent. Mutations in PIK3CA were present in 65 samples (43.6%) which is similar to that reported in TCGA database. PIK3R1 (37%) was found significantly mutated, with a novel recurrent mutation, N595S, being identified in 24 patients. Similarly, AKT1 (10.1%), AKT2 (10.1%), and AKT3 (14.8%) mutations were present at a higher frequency in our population than has been reported in the TCGA and COSMIC database. In the PIK3CA-GaMB library, our highest-ranking mutations included the previously validated deleterious mutations H1047R and E545K and several mutations of uncertain significance, including E39K, G1049R, N345I, N345K, M1043V, and H1047T. In the validation assays, we found a high phenotype-consistency of these identified mutations using these functional validation techniques. The breast cancer cells harboring identified mutations exhibit a relatively higher proliferation ability and tolerance to chemotherapy and pathway inhibitors. Conclusion This study identified the landscape of genetic mutations in the PI3K/AKT pathway using Amplicon Sequencing in a Chinese population. A novel developed GaMB screening platform may allow the rapid identification of significant mutations that dominate breast cancer development and drug responses during treatment. Citation Format: Chen L, Yang L, Yao L, Hu X, Shao Z. The mutation detection and a high throughput screening of driver mutations in PI3K/AKT pathway based on next generation sequencing [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-13.

Rapid and accurate identification of SNPs and mutations are useful in ensuring that the correctly identified cell lines are being used in cancer research studies and for accurate identification of SNPs and indels in cancer cells. In this study we report on a method that uses nucleic acid isolation methods, multiplexed PCR to amplify up to 48 regions within the human genome followed by an oligonucleotide ligation assay that can detect SNPs, mutations and indels. The protocol takes about 4 hrs to complete and uses panels optimized for human cell identification and detection of mutations in EGFR and KRAS genes. We typed the NCI-60 panel and several cell lines from the ATCC using two panels. One is a panel of SNPs that has been tested with samples from over 46 human populations to identify a set of SNPs that would discriminate globally. This SNP set has 48 markers with high heterozygosity (>0.4) and low Fst values (<0.06) across multiple human populations. The panel has a high power of discrimination. (Hum Genet; 127 (3) 315-324; 2010) comparable to that obtained with STR kits. A panel of 31 samples from closely related individuals within families was typed to test the power of discrimination. In another study, we applied this system to a cancer mutation study, in which an assay for detecting 29 EGFR and 5 KRAS gene mutations was developed. These mutations are frequently found in human cancers. DNA preparations from 48 ATCC and 60 NCI tumor cell lines were genotyped by this assay. These SNPs and mutations were further confirmed by the Sanger sequencing chemistry, showing 100% concordance between two technologies. The information generated from this study will be definitely useful when these cell lines are used as models for cancer study and cancer drug screening. In conclusion, we have developed a sensitive, highly accurate, high throughput genotyping system. Studies have demonstrated its effectiveness for successful SNP/InDel detection and mutation screening. This system has been successfully used for oncogen mutation studies in cancer cell lines. Overall, the system can become a valuable tool for molecular and clinical diagnostics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3883. doi:10.1158/1538-7445.AM2011-3883

Identification of mutations in circulating cell-free tumour DNA as a biomarker in hepatocellular carcinoma

Immunohistochemistry as a reliable method for detection of BRAF-V600E mutation in melanoma: a systematic review and meta-analysis of current published literature