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1.
Biotechniques ; 67(1): 6-10, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31124706

RESUMO

SNPs and single base pair (SBP) insertion/deletions (indels) are not only the most abundant genetic markers for genetic mapping and breeding selection, but also always occur in the mutants generated from chemical mutagenesis or CRISPR/Cas9-mediated genome editing. Most of the current SNP and SBP indel genotyping methods are time-consuming and/or require special equipment or reagents. Here, we describe an improved heteroduplex analysis method, named iHDA, that can readily discriminate SNP and SBP indel alleles with specially designed DNA probes that harbor a couple of nucleotides adjacent to the SNP site. By hybridizing with the same probe, SNP and SBP indel alleles form different heteroduplexes, differing in bulge size, which show different mobility on a polyacrylamide gel. Therefore, iHDA is an easy, fast and inexpensive method for SNP and SBP indel genotyping.


Assuntos
Análise Heteroduplex/métodos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Pareamento de Bases , Sistemas CRISPR-Cas , DNA de Plantas/genética , Técnicas de Genotipagem/economia , Técnicas de Genotipagem/métodos , Análise Heteroduplex/economia , Oryza/genética , Fatores de Tempo
2.
Methods Mol Biol ; 1956: 77-103, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30779031

RESUMO

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) or consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium. The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan and heteroduplex analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.


Assuntos
Rearranjo Gênico , Análise Heteroduplex/métodos , Transtornos Linfoproliferativos/genética , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Sequência de Bases , Células Clonais/metabolismo , Células Clonais/patologia , DNA/genética , DNA/isolamento & purificação , Genes Codificadores dos Receptores de Linfócitos T , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulinas/genética , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia
3.
Methods Mol Biol ; 1469: 123-35, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27557690

RESUMO

Sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system, are powerful tools for understanding gene function and for developing novel traits in plants. In plant species for which transformation and regeneration systems using protoplasts are not yet established, direct delivery to nuclei of SSNs either in the form of RNA or protein is difficult. Thus, Agrobacterium-mediated transformation of SSN expression constructs in cultured cells is a practical means of delivering targeted mutagenesis in some plant species including rice. Because targeted mutagenesis occurs stochastically in transgenic cells and SSN-mediated targeted mutagenesis often leads to no selectable phenotype, identification of highly mutated cell lines is a critical step in obtaining regenerated plants with desired mutations.


Assuntos
Sistemas CRISPR-Cas , Mutagênese Sítio-Dirigida/métodos , Oryza/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Agrobacterium/genética , Vetores Genéticos , Análise Heteroduplex , Mutagênese , Taxa de Mutação , Plantas Geneticamente Modificadas
4.
Biochem Med (Zagreb) ; 26(2): 264-71, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27346974

RESUMO

Congenital analbuminemia is an autosomal recessive disorder, in which albumin, the major blood protein, is present only in a minute amount. The condition is a rare allelic heterogeneous defect, only about seventy cases have been reported worldwide. To date, more than twenty different mutations within the albumin gene have been found to cause the trait. In our continuing study of the molecular genetics of congenital analbuminemia, we report here the clinical and biochemical findings and the mutation analysis of the gene in two Turkish infants. For the molecular analysis, we used our strategy, based on the screening of the gene by single-strand conformation polymorphism, heteroduplex analysis and direct DNA sequencing. The results showed that both patients are homozygous for the deletion of a cytosine residue in exon 5, in a stretch of four cytosines starting from nucleotide position 524 and ending at position 527 (NM_000477.5(ALB):c.527delC). The subsequent frame-shift inserts a stop codon in position 215, markedly reducing the size of the predicted protein product. The parents are both heterozygous for the same mutation, for which we propose the name Erzurum from the city of origin of the family. In conclusion, our results show that in this family congenital analbuminemia is caused by a novel frame-shift/deletion defect, confirm the inheritance of the trait, and contribute to advance our understanding of the molecular basis underlying this condition.


Assuntos
Mutação da Fase de Leitura/genética , Deleção de Sequência/genética , Albumina Sérica/deficiência , Albumina Sérica/genética , Adulto , Feminino , Análise Heteroduplex , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Lactente , Masculino , Linhagem , Turquia
5.
MEDICC Rev ; 17(4): 25-31, 2015 10.
Artigo em Inglês | MEDLINE | ID: mdl-26947278

RESUMO

INTRODUCTION Serological and molecular HIV-1 studies in Cuba have shown very low prevalence of seropositivity, but an increasing genetic diversity attributable to introduction of many HIV-1 variants from different areas, exchange of such variants among HIV-positive people with several coinciding routes of infection and other epidemiologic risk factors in the seropositive population. The high HIV-1 genetic variability observed in Cuba has possible implications for transmission and clinical progression. OBJECTIVE Study genetic variability for the HIV-1 env, gag and pol structural genes in Cuba; determine the prevalence of B and non-B subtypes according to epidemiologic and behavioral variables and determine whether a relationship exists between genetic variability and transmissibility, and between genetic variability and clinical disease progression in people living with HIV/AIDS. METHODS Using two molecular assays (heteroduplex mobility assay and nucleic acid sequencing), structural genes were characterized in 590 people with HIV-1 (480 men and 110 women), accounting for 3.4% of seropositive individuals in Cuba as of December 31, 2013. Nonrandom sampling, proportional to HIV prevalence by province, was conducted. Relationships between molecular results and viral factors, host characteristics, and patients' clinical, epidemiologic and behavioral variables were studied for molecular epidemiology, transmission, and progression analyses. RESULTS Molecular analysis of the three HIV-1 structural genes classified 297 samples as subtype B (50.3%), 269 as non-B subtypes (45.6%) and 24 were not typeable. Subtype B prevailed overall and in men, mainly in those who have sex with men. Non-B subtypes were prevalent in women and heterosexual men, showing multiple circulating variants and recombinant forms. Sexual transmission was the predominant form of infection for all. B and non-B subtypes were encountered throughout Cuba. No association was found between subtypes and transmission or clinical progression, although the proportion of deaths was higher for subtype B. Among those who died during the study period, there were no differences between subtypes in the mean time from HIV or AIDS diagnosis to death. CONCLUSIONS Our results suggest that B and non-B HIV-1 subtypes found in Cuba do not differ in transmissibility and in clinical disease progression. KEYWORDS HIV-1, AIDS, molecular epidemiology, transmissibility, clinical progression, subtypes, circulating recombinant forms, pathogenesis, Cuba.


Assuntos
Variação Genética , Infecções por HIV/genética , Infecções por HIV/transmissão , HIV-1/genética , Adolescente , Adulto , Idoso , Cuba/epidemiologia , Progressão da Doença , Feminino , Infecções por HIV/epidemiologia , Análise Heteroduplex , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Replicação de Sequência Autossustentável
6.
Methods Mol Biol ; 1245: 141-50, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25373754

RESUMO

Heteroduplex-based genotyping methods have proven to be technologically effective and economically efficient for low- to medium-range throughput single-nucleotide polymorphism (SNP) determination. In this chapter we describe two protocols that were successfully applied for SNP detection and haplotype analysis of candidate genes in association studies. The protocols involve (1) enzymatic mismatch cleavage with endonuclease CEL1 from celery, associated with fragment separation using capillary electrophoresis (CEL1 cleavage), and (2) differential retention of the homo/heteroduplex DNA molecules under partial denaturing conditions on ion pair reversed-phase liquid chromatography (dHPLC). Both methods are complementary since dHPLC is more versatile than CEL1 cleavage for identifying multiple SNP per target region, and the latter is easily optimized for sequences with fewer SNPs or small insertion/deletion polymorphisms. Besides, CEL1 cleavage is a powerful method to localize the position of the mutation when fragment resolution is done using capillary electrophoresis.


Assuntos
Técnicas de Genotipagem/métodos , Análise Heteroduplex/métodos , Polimorfismo de Nucleotídeo Único/genética , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Endonucleases/metabolismo , Genótipo , Helianthus/genética , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico
7.
J Clin Microbiol ; 53(2): 597-610, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25502529

RESUMO

Key clinical studies for HIV coreceptor antagonists have used the phenotyping-based Trofile test. Meanwhile various simpler-to-do genotypic tests have become available that are compatible with standard laboratory equipment and Web-based interpretation tools. However, these systems typically analyze only the most prominent virus sequence in a specimen. We present a new diagnostic HIV tropism test not needing DNA sequencing. The system, XTrack, uses physical properties of DNA duplexes after hybridization of single-stranded HIV-1 env V3 loop probes to the clinical specimen. Resulting "heteroduplexes" possess unique properties driven by sequence relatedness to the reference and resulting in a discrete electrophoretic mobility. A detailed optimization process identified diagnostic probe candidates relating best to a large number of HIV-1 sequences with known tropism. From over 500 V3 sequences representing all main HIV-1 subtypes (Los Alamos database), we obtained a small set of probes to determine the tropism in clinical samples. We found a high concordance with the commercial TrofileES test (84.9%) and the Web-based tool Geno2Pheno (83.0%). Moreover, the new system reveals mixed virus populations, and it was successful on specimens with low virus loads or on provirus from leukocytes. A replicative phenotyping system was used for validation. Our data show that the XTrack test is favorably suitable for routine diagnostics. It detects and dissects mixed virus populations and viral minorities; samples with viral loads (VL) of <200 copies/ml are successfully analyzed. We further expect that the principles of the platform can be adapted also to other sequence-divergent pathogens, such as hepatitis B and C viruses.


Assuntos
Técnicas de Genotipagem/métodos , Infecções por HIV/diagnóstico , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/fisiologia , Tropismo Viral , Estudos de Coortes , Ensaio de Desvio de Mobilidade Eletroforética , HIV-1/genética , Análise Heteroduplex , Humanos , Hibridização de Ácido Nucleico
8.
ACS Chem Biol ; 9(9): 1975-9, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-25050989

RESUMO

We describe the effects of introducing two epimers of neutral backbone α,ß-constrained nucleic acid (CNA) on the activity and allele selectivity profile of RNase H active antisense oligonucleotides (ASOs) targeting a single nucleotide polymorphism (SNP) for the treatment of Huntington's disease (HD). ASOs modified with both isomers of α,ß-CNA in the gap region showed good activity versus the mutant allele, but one isomer showed improved selectivity versus the wild-type allele. Analysis of the human RNase H cleavage patterns of α,ß-CNA modified ASOs versus matched and mismatched RNA revealed that both isomers support RNase H cleavage on the RNA strand across from the site of incorporation in the ASO--an unusual observation for a neutral linkage oligonucleotide modification. Interestingly, ASOs modified with (R)- and (S)-5'-hydroxyethyl DNA (RHE and SHE respectively) formed by partial hydrolysis of the dioxaphosphorinane ring system in α,ß-CNA also showed good activity versus the mutant allele but an improved selectivity profile was observed for the RHE modified ASO. Our observations further support the profiling of neutral and 5'-modified nucleic acid analogs as tools for gene silencing applications.


Assuntos
Proteínas do Tecido Nervoso/genética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Polimorfismo de Nucleotídeo Único , Alelos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Inativação Gênica , Análise Heteroduplex , Humanos , Proteína Huntingtina , Hidrólise , Terapia de Alvo Molecular , Mutação , Proteínas do Tecido Nervoso/metabolismo , Nucleotídeos/química , RNA/química , Ribonuclease H/metabolismo , Estereoisomerismo
9.
Biochem Med (Zagreb) ; 24(1): 151-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24627724

RESUMO

INTRODUCTION: Congenital analbuminemia is a rare autosomal recessive disorder manifested by the presence of a very low amount of circulating serum albumin. It is an allelic heterogeneous defect, caused by variety of mutations within the albumin gene in homozygous or compound heterozygous state. Herein we report the clinical and molecular characterization of a new case of congenital analbuminemia diagnosed in a female newborn of consanguineous (first degree cousins) parents from Ankara, Turkey, who presented with a low albumin concentration (< 8 g/L) and severe clinical symptoms. MATERIALS AND METHODS: The albumin gene of the index case was screened by single-strand conformation polymorphism, heteroduplex analysis, and direct DNA sequencing. The effect of the splicing mutation was evaluated by examining the cDNA obtained by reverse transcriptase - polymerase chain reaction (RT-PCR) from the albumin mRNA extracted from proband's leukocytes. RESULTS: DNA sequencing revealed that the proband is homozygous, and both parents are heterozygous, for a novel G>A transition at position c.1652+1, the first base of intron 12, which inactivates the strongly conserved GT dinucleotide at the 5' splice site consensus sequence of this intron. The splicing defect results in the complete skipping of the preceding exon (exon 12) and in a frame-shift within exon 13 with a premature stop codon after the translation of three mutant amino acid residues. CONCLUSIONS: Our results confirm the clinical diagnosis of congenital analbuminemia in the proband and the inheritance of the trait and contribute to shed light on the molecular genetics of analbuminemia.


Assuntos
Processamento Alternativo/genética , Hipoalbuminemia/congênito , Hipoalbuminemia/genética , Mutação/genética , Albumina Sérica/genética , Adolescente , Adulto , Sequência de Bases , Feminino , Análise Heteroduplex , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Polimorfismo Conformacional de Fita Simples , Albumina Sérica/deficiência , Albumina Sérica Humana , Adulto Jovem
10.
J Clin Microbiol ; 52(1): 298-301, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24131693

RESUMO

A heteroduplex tracking assay used to genotype Plasmodium vivax merozoite surface protein 1 was adapted to a capillary electrophoresis format, obviating the need for radiolabeled probes and allowing its use in settings where malaria is endemic. This new assay achieved good allelic discrimination and detected high multiplicities of infection in 63 P. vivax infections in Cambodia. More than half of the recurrent parasitemias sampled displayed identical or highly related genotypes compared to the initial genotype, suggesting that they represented relapses.


Assuntos
Eletroforese Capilar/métodos , Variação Genética , Análise Heteroduplex/métodos , Malária Vivax/parasitologia , Proteína 1 de Superfície de Merozoito/genética , Plasmodium vivax/classificação , Plasmodium vivax/genética , Camboja , DNA de Protozoário/química , DNA de Protozoário/genética , Humanos , Dados de Sequência Molecular , Plasmodium vivax/isolamento & purificação , Recidiva , Análise de Sequência de DNA
11.
Dev Growth Differ ; 56(1): 98-107, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24286287

RESUMO

Genome editing using engineered nucleases such as transcription activator-like effector nucleases (TALENs) has become a powerful technology for reverse genetics. In this study, we have described efficient detection methods for TALEN-induced mutations at endogenous loci and presented guidelines of TALEN design for efficient targeted mutagenesis in medaka, Oryzias latipes. We performed a heteroduplex mobility assay (HMA) using an automated microchip electrophoresis system, which is a simple and high-throughput method for evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or later generations. We found that a specific pattern of mutations is dominant for TALENs harboring several base pairs of homologous sequences in target sequence. Furthermore, we found that a 5' T, upstream of each TALEN-binding sequence, is not essential for genomic DNA cleavage. Our findings provide information that expands the potential of TALENs and other engineered nucleases as tools for targeted genome editing in a wide range of organisms, including medaka.


Assuntos
Endodesoxirribonucleases/metabolismo , Marcação de Genes/métodos , Mutagênese Sítio-Dirigida/métodos , Oryzias/genética , Animais , Endodesoxirribonucleases/genética , Análise Heteroduplex
12.
Acta Cytol ; 57(6): 646-51, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24107477

RESUMO

OBJECTIVE: Keratoconus (KC) is an eye disorder in which the cornea is swollen, thinned and deformed. Despite extensive studies, the pathophysiological processes and genetic etiology of KC are unknown. The disease incidence is approximately 1 in 2,000, and it is the most common cause of corneal transplantation in the USA. Many genes are involved in the disease, but evidence suggests a major role for VSX1 in the etiology of KC. This study aimed to determine the frequency of mutations in exons 2, 3 and 4 of the VSX1 gene in Chaharmahal va Bakhtiari province in the southwest of Iran. STUDY DESIGN: In this experimental study, mutations in 3 exons, namely exons 2, 3 and 4, of VSX1 were investigated in 50 patients with KC and 50 healthy control subjects. DNA was extracted using a standard phenol-chloroform method. PCR-single-strand conformational polymorphism/heteroduplex analysis was performed, followed by DNA sequencing to confirm the identified motility shifts. RESULTS: H244R mutations were found in 1 patient and also in 1 healthy control subject. Furthermore, 12 polymorphisms were identified in patients with KC and 7 in healthy control subjects [rs6138482 and c.546A>G (rs12480307)]. CONCLUSION: Our investigation showed that KC-related VSX1 mutations were found in a very small proportion of the studied patients from Iran. Further investigations on other genes are needed to clarify their roles in KC pathogenesis.


Assuntos
Proteínas do Olho/genética , Análise Heteroduplex/métodos , Proteínas de Homeodomínio/genética , Ceratocone/genética , Mutação , Sequência de Bases , Análise Mutacional de DNA , Humanos , Irã (Geográfico) , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Polimorfismo Conformacional de Fita Simples
13.
J Virol Methods ; 194(1-2): 169-77, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23994080

RESUMO

Heteroduplex mobility (HMA) and tracking assays (HTA) are used to assess genetic relationships between DNA molecules. While distinguishing relationships between clonal or nearly clonal molecules is relatively straightforward, inferring population structures is more complex. To address this issue, HIV-1 quasispecies with varying levels of diversity were studied using both HTA and DNA sequencing. Viral diversity estimates and the temporal features of virus evolution were found to be generally concordant between HTA and DNA sequencing. In addition, the distribution of pairwise differences and the rates of virus divergence were similar between the two methods. These findings support the use of HTA to characterize variant populations of DNA and strengthen previous inferences concerning the evolution of HIV-1 over the course of infection.


Assuntos
Variação Genética , HIV-1/classificação , HIV-1/genética , Análise Heteroduplex/métodos , Análise de Sequência de DNA/métodos , Virologia/métodos , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Humanos
14.
DNA Cell Biol ; 32(11): 640-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24024466

RESUMO

There is evidence of linkage between the 15q13-q14 locus, containing the gene encoding the α7 subunit (CHRNA7) of the neuronal nicotinic acetylcholine receptor (nAChR) and its partially duplicated isoform (CHRFAM7A), and epilepsy. Additionally, a 2-bp deletion polymorphism (c.497-498delTG; rs67158670) in CHRFAM7A, resulting in a frame shift and truncation of the protein product, is associated with some neurological diseases. This study was designed to explore the possibility of an association of the c.497-498delTG polymorphism of CHRFAM7A with idiopathic generalized epilepsies (IGEs) in Polish children and young patients. The study included 197 IGE patients and 258 unrelated healthy individuals. The frequency of the CHRFAM7A c.497-498delTG polymorphism was determined in each group using heteroduplex analysis. An association between the c.497-498delTG polymorphism of CHRFAM7A and IGE was evidenced. It was demonstrated that the frequency of the CHRFAM7A 2-bp deletion carriers was significantly lower in the IGE patients than in the control group. The observed frequency of 2-bp deletion carriers was high in IGE subjects (64%), but significantly higher in control subjects (76%). Carriers of at least one copy of the -2 bp allele had halved their risk of IGE susceptibility (delTG/delTG and delTG/wild-type versus wild-type/wild-type: odds ratio=0.55; 95% confidence intervals=0.365-0.827; p=0.004). Moreover, it has been demonstrated that this polymorphic variant is associated with the c.524-12_524-11insGTT variation (rs10649395) in intron 7 of CHRFAM7A. Our study substantiates the involvement of the α7 subunit of nAChR in the pathophysiology of IGEs and indicates that the CHRFAM7A c.497-498TG deletion or a nearby polymorphism may play a role in the pathogenesis of IGE. Further work should concentrate on ascertaining the exact mechanism of this polymorphism's effect and its relationship with IGE.


Assuntos
Epilepsia Generalizada/genética , Polimorfismo Genético , Receptor Nicotínico de Acetilcolina alfa7/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Intervalos de Confiança , Feminino , Frequência do Gene , Predisposição Genética para Doença , Análise Heteroduplex , Humanos , Íntrons , Masculino , Polônia , Deleção de Sequência , Adulto Jovem
15.
J Am Mosq Control Assoc ; 29(2): 102-7, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23923324

RESUMO

Mortality of American white pelican (Pelecanus erythrorhynchos) chicks attributed to West Nile virus (WNV) prompted field studies on the bionomics of mosquitoes on a wildlife refuge in northern Montana. One component of these studies was to identify blood meal sources for Culex tarsalis, the primary vector of WNV in the region, and the potential bridge vectors Aedes vexans and Culiseta inornata. To accomplish this, 3 methods were evaluated to collect bloodfed mosquitoes: a gasoline powered aspirator, CO2-baited light traps, and fiber pots in shelterbelts consisting of stands of deciduous trees and shrubs and marshes along the lake edge. Fiber pots were also deployed in open fields of prairie grasses. Overall, fiber pots were the most efficient method for collecting engorged Cx. tarsalis and Cs. inornata, largely due to shorter sampling and processing times. Aedes vexans was not collected in fiber pots but was more abundant in aspiration samples than the other 2 species. The optimal location for collecting Cx. tarsalis was dependent on trapping method. Aspirations and fiber pot placements collected more Cx. tarsalis in shelterbelts, while CO2-baited light traps collected more Cx. tarsalis in the marsh habitat. Sixteen avian and 4 mammalian hosts were identified from bloodfed Cx. tarsalis with 46 blood meals derived from birds and 49 from mammals. Aedes vexans and Cs. inornata fed predominantly on white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus), respectively. Humans were identified as hosts in 33% of engorged Cx. tarsalis, 4% of engorged Ae. vexans, and 18% of engorged Cs. inornata.


Assuntos
Culicidae/fisiologia , Insetos Vetores/fisiologia , Controle de Mosquitos/métodos , Animais , Aves/fisiologia , Culicidae/classificação , Ecossistema , Comportamento Alimentar , Feminino , Cadeia Alimentar , Análise Heteroduplex/veterinária , Insetos Vetores/classificação , Masculino , Mamíferos/fisiologia , Montana/epidemiologia , Controle de Mosquitos/instrumentação , Reação em Cadeia da Polimerase/veterinária , Especificidade da Espécie , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/fisiologia
16.
Methods Mol Biol ; 1015: 25-54, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23824847

RESUMO

Denaturing high-performance liquid chromatography (DHPLC) is an accurate and efficient screening technique used for detecting DNA sequence changes by heteroduplex analysis. It can also be used for genotyping of single nucleotide polymorphisms (SNPs). The high sensitivity of DHPLC has made this technique one of the most reliable approaches to mutation analysis and, therefore, used in various areas of genetics, both in the research and clinical arena. This chapter describes the methods used for mutation detection analysis and the genotyping of SNPs by DHPLC on the WAVE™ system from Transgenomic Inc. ("WAVE" and "DNASep" are registered trademarks, and "Navigator" is a trademark, of Transgenomic, used with permission. All other trademarks are property of the respective owners).


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Análise Mutacional de DNA/métodos , Técnicas de Genotipagem/métodos , Análise Heteroduplex/métodos , Humanos , Mutação , Desnaturação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único
17.
Genes Cells ; 18(6): 450-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23573916

RESUMO

The heteroduplex mobility assay (HMA) is widely used to characterize strain variants of human viruses. To determine whether it can detect small sequence differences in homologous templates, we constructed a series of deletion constructs (1-10 bp deletions) in the multiple cloning site (MCS) of pBluescript II. After PCR amplification of the MCS using a mixture of wild-type and one of the deletion constructs, the resulting PCR amplicons were electrophoresed using 15% polyacrylamide gels. Two types of heteroduplexes exhibited retarded electrophoretic migration compared with individual homoduplexes. Therefore, we applied this HMA to detect transcription activator-like effector nucleases (TALEN)-induced insertion and/or deletion (indel) mutations at an endogenous locus. We found that TALEN in vivo activity was easily estimated by the degree of multiple HMA profiles derived from TALEN-injected F0 embryos. Furthermore, TALEN-injected F0 founder fish produced several unique HMA profiles in F1 embryos. Sequence analysis confirmed that the different HMA profiles contained distinct indel mutations. Thus, HMA is a rapid and sensitive analytical method for the detection of the TALEN-mediated genome modifications.


Assuntos
Desoxirribonucleases/metabolismo , Genoma/genética , Análise Heteroduplex , Peixe-Zebra/genética , Animais , Mutação INDEL/genética
18.
Am J Trop Med Hyg ; 88(6): 1116-23, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23509126

RESUMO

Plasmodium vivax accounts for an increasing fraction of malaria infections in Thailand and Cambodia. We compared P. vivax genetic complexity and antimalarial resistance patterns in the two countries. Use of a heteroduplex tracking assay targeting the merozoite surface protein 1 gene revealed that vivax infections in both countries are frequently polyclonal (84%), with parasites that are highly diverse (HE = 0.86) but closely related (GST = 0.18). Following a history of different drug policies in Thailand and Cambodia, distinct patterns of antimalarial resistance have emerged: most Cambodian isolates harbor the P. vivax multidrug resistance gene 1 (pvmdr1) 976F mutation associated with chloroquine resistance (89% versus 8%, P < 0.001), whereas Thai isolates more often display increased pvmdr1 copy number (39% versus 4%, P < 0.001). Finally, genotyping of paired isolates from individuals suspected of suffering relapse supports a complex scheme of relapse whereby recurrence of multiple identical variants is sometimes accompanied by the appearance of novel variants.


Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Alelos , Antimaláricos/uso terapêutico , Biodiversidade , Camboja , Cloroquina/uso terapêutico , Resistência a Medicamentos , Dosagem de Genes , Genótipo , Análise Heteroduplex , Humanos , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNA , Tailândia
19.
Methods Mol Biol ; 971: 65-91, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296958

RESUMO

The assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes is a valuable technique in the diagnosis of suspect lymphoproliferative disorders. Furthermore this technique is more and more used to evaluate dissemination of non-Hodgkin lymphoma and/or the presence of (minimal) residual disease. In this chapter we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TCRB), and TCR gamma (TCRG) gene rearrangements. The described PCR protocol is based on the standardized multiplex PCRs as developed by the European BIOMED-2 collaborative study (Concerted Action BMH4-CT98-3936). Furthermore it also includes the pre-analytical DNA isolation step from various tissues (formalin fixed paraffin-embedded tissue, fresh tissues, body fluids, peripheral blood and bone marrow), GeneScan analysis of labeled PCR products on a genetic analyzer, heteroduplex analysis of unlabeled PCR products, and post-analytical guidelines for the interpretation of the obtained "molecular morphology" patterns.


Assuntos
Rearranjo Gênico/genética , Análise Heteroduplex/métodos , Imunoglobulinas/genética , Linfoma/diagnóstico , Linfoma/genética , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Células da Medula Óssea/patologia , Proliferação de Células , Células Clonais/metabolismo , Células Clonais/patologia , DNA/genética , DNA/isolamento & purificação , Formaldeído/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Linfoma/sangue , Linfoma/patologia , Inclusão em Parafina , Linfócitos T/metabolismo , Linfócitos T/patologia , Fixação de Tecidos
20.
Methods Mol Biol ; 971: 175-200, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296964

RESUMO

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell Non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is the gold-standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). RQ-PCR analysis of the junctional regions of the rearranged immunoglobulin heavy-chain gene (IgH) serves as the most broadly applicable MRD target in B-NHL (∼80%). Chromosomal translocations as t(14;18) translocation in FL and t(11;14) translocation in MCL can be used in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry as well as RQ-PCR are equally suitable for MRD assessment as long as a sensitivity of ≤10(-4) shall be achieved.MRD diagnostics targeting the IgH gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. Furthermore, somatic mutations of the IgH region occurring during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IgH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10(-4)). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IgH gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.


Assuntos
Rearranjo Gênico/genética , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Células B/genética , Linfoma de Células B/patologia , Neoplasia Residual/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Translocação Genética/genética , Alelos , Células da Medula Óssea/patologia , Primers do DNA/genética , Análise Heteroduplex , Humanos , Leucócitos Mononucleares/patologia , Oligodesoxirribonucleotídeos/genética , Análise de Sequência de DNA
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