RESUMO
Folding primer (FP), together with turn-back primer (TP) and boost primer (BP), is one of the major components of SmartAmp2, a rapid amplification-based method for SNP detection. FP has a unique design where the annealing region is combined with a tail that can fold back. FP tails can be classified as either "strong" or "weak", depending on the melting temperature and free energy of the hairpin structure. We report that FP tails affect the amplification process differently; by changing the FP concentration, we can increase the amplification reaction speed with "strong tails". Unlike "strong tails", concentration change of FP with "weak tails" did not show significant impact on the amplification speed. The comparative analyses using gel electrophoresis demonstrate that the FP type and FP ratio in the reaction change the amplification pattern. The above observations can be used to optimize the reaction and manipulate the reaction speed of SmartAmp2.
Assuntos
Primers do DNA/química , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , DNA/análise , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Conformação de Ácido Nucleico , Sensibilidade e Especificidade , Temperatura de TransiçãoRESUMO
BACKGROUND: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. RESULTS: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.
Assuntos
Hidrocarboneto de Aril Hidroxilases/análise , Hidrocarboneto de Aril Hidroxilases/genética , Oxigenases de Função Mista/análise , Oxigenases de Função Mista/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Polimorfismo de Nucleotídeo Único/genética , Varfarina/farmacologia , Hidrocarboneto de Aril Hidroxilases/classificação , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sequência de Bases , Citocromo P-450 CYP2C9 , Relação Dose-Resposta a Droga , Humanos , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Fatores de Tempo , Vitamina K Epóxido RedutasesRESUMO
Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.
Assuntos
Análise Mutacional de DNA/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Mutação Puntual , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de DNA/métodos , Proteínas ras/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Análise Mutacional de DNA/instrumentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Proteínas Proto-Oncogênicas p21(ras) , Sensibilidade e Especificidade , Análise de Sequência de DNA/instrumentaçãoRESUMO
A key feature of the smart amplification process version 2 (SMAP-2) is the ability to suppress mismatch amplification by using a unique asymmetric primer design and Thermus aquaticus MutS (Taq MutS). However we report here that use of SMAP-2 for polymorphism determination of the UGT1A1 *28 allele required a further ancillary approach for complete background suppression. The UGT1A1 *28 allele is a microsatellite copy number polymorphism. This is the first reported SMAP-2 assay designed for genotyping genetic variations of microsatellites. We found that by the addition of a primer to the amplification reaction, called a competitive probe (CP), assay specificity could be significantly enhanced. Including sample preparation time and use of a CP-enhanced SMAP-2 assay, we could rapidly detect the UGT1A1 *28 polymorphism within 60 min. To test our method, we compared results from PCR sequencing and the CP-enhanced SMAP-2 assay on 116 human blood samples for UGT1A1 *28 polymorphism and demonstrated perfect concordance. These results illustrate the versatility of SMAP-2 for molecular diagnostics and provide a new approach for enhancing SMAP-2 assay specificity.
Assuntos
Bioensaio/métodos , Sondas de DNA/genética , Glucuronosiltransferase/genética , Repetições de Microssatélites/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA/métodos , Genótipo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Gene expression profiles in the amygdala of juvenile rats were compared between the two autistic rat models for mechanistic insights into impaired social behavior and enhanced anxiety in autism. The rats exposed to VPA by intraperitoneal administration to their dams at embryonic day (E) 12 were used as a model for autism (E2IP), and those by subcutaneous administration at postnatal day (P) 14 (P14SC) were used as a model for regressive autism; both of the models show impaired social behavior and enhanced anxiety as symptoms. Gene expression profiles in the amygdala of the rats (E12IP and P14SC) were analyzed by microarray and compared to each other. Only two genes, Neu2 and Mt2a, showed significant changes in the same direction in both of the rat models, and there were little similarities in the overall gene expression profiles between them. It was considered that gene expression changes per se in the amygdala might be an important cause for impaired social behavior and enhanced anxiety, rather than expression changes of particular genes.
Assuntos
Tonsila do Cerebelo , Transtorno Autístico/genética , Metalotioneína/genética , Metalotioneína/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo , Transcriptoma/genética , Ácido Valproico/toxicidade , Animais , Animais Recém-Nascidos , Ansiedade/genética , Transtorno Autístico/psicologia , Comportamento Animal , Modelos Animais de Doenças , Feminino , Injeções Intraperitoneais , Injeções Subcutâneas , Exposição Materna , Troca Materno-Fetal , Gravidez , Ratos , Ratos Wistar , Comportamento Social , Ácido Valproico/administração & dosagem , Ácido Valproico/metabolismoRESUMO
BACKGROUND: In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society. METHODOLOGY: To address the clinical need for rapid diagnosis, we have developed a new method, the "RT-SmartAmp assay", to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses. RESULTS AND CONCLUSIONS: We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/epidemiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , DNA Polimerase Dirigida por RNA/metabolismo , Idoso , Criança , Primers do DNA/genética , Farmacorresistência Viral , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Oseltamivir/farmacologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Fatores de TempoRESUMO
We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.