RESUMEN
A panel of 106 insertion/deletion (InDel) polymorphisms and a method of their genotyping on biochips were proposed as a new approach to genetic personal identification. Short lengths and low mutation rates are basic properties of InDel markers, which thus have significant advantages over short tandem repeats (STRs) widely used in forensics. The allele frequency distributions of all known InDel polymorphisms were studied in the five largest world populations (European, East Asian, South Asian, African, and American). Markers were selected to meet the following criteria: the minor allele frequency (MAF) is higher than 0.30; the physical distance between markers is greater than 3 Mb; there are no polymorphisms, tandem repeats, and palindromes in the flanking sequences; the AT/GC ratio is close to 1. A panel of 106 polymorphisms was thus formed; the average MAF was estimated at 0.396 in the five populations. The method developed for panel genotyping included one-step multiplex PCR and subsequent hybridization on a biological microarray. The average amplicon length was 72 bp. A sample of 201 residents of Moscow and St. Petersburg was tested to determine the main characteristics of the panel: the random matching probability (MP) was 1.89x 10^(-43) and the combined probability of paternity exclusion (CPE) was 0.99999999063. The method provides an alternative to molecular genetic personal identification based on the STR length variations.
Asunto(s)
Genética de Población , Mutación INDEL , Polimorfismo Genético , Humanos , Frecuencia de los Genes , Repeticiones de MicrosatéliteRESUMEN
This paper presents a method for genotyping a panel of 60 single nucleotide polymorphisms (SNPs) using single-stage PCR followed by hybridization on a hydrogel biochip. The pool of analyzed polymorphisms consists of 41 SNPs included in the HIrisPlex-S panel, 4 SNPs of the AB0 gene (261G>Del, 297A>G, 657C>T, 681G>A), markers of the AMELX and AMELY genes, and 14 SNP markers of the Y chromosome haplogroups: B (M60), C (M130), D (CTS3946), E (M5388), G (P257), H (M2920), I (U179), J (M304), L (M185), N (M231), O (M175), Q (M1105), R (P224) and T (M272). These genetic data allow one to predict the phenotype of the desired person according to the characteristics of eye, hair, skin color, AB0 blood group, sex, and genogeographic origin in the male line. The setting protocol is simplified as much as possible to facilitate the introduction of the method into practice. The distribution of allele frequencies of the studied polymorphisms, as well as AB0 blood groups among the Slavs (N = 482), originating mainly from central Russia, was established.
Asunto(s)
Sistema del Grupo Sanguíneo ABO , Cromosomas Humanos Y , Color del Ojo , Técnicas de Genotipaje , Color del Cabello , Análisis de Secuencia por Matrices de Oligonucleótidos , Pigmentación de la Piel , Sistema del Grupo Sanguíneo ABO/genética , Cromosomas Humanos Y/genética , Color del Ojo/genética , Color del Cabello/genética , Haplotipos , Humanos , Hidrogeles , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple , Pigmentación de la Piel/genética , Población Blanca/genéticaRESUMEN
The substrate properties of nitrogen-base modified derivatives of purine and pyrimidine deoxynucleoside triphosphates during their simultaneous pairwise insertion into the growing DNA strand have been studied. Modified nucleotides were introduced using real-time PCR and the primer extension reaction; in one reaction, derivatives with both different and similar functional substituents were used. Genomic bacterial DNA, specially constructed synthetic DNA fragments, and SELEX libraries were used as templates. The reactions were performed using DNA polymerases with no 3'-5' correcting exonuclease activity: Taq, Vent (exo-), DeepVent (exo-), and KOD XL. It was shown that the substrate efficiency is affected by both the size of the substituent group and the chemical nature of deoxynucleoside triphosphate. The effectiveness varies significantly depending on the polymerase used. The most effective of the studied substrates are pyrimidine deoxynucleoside triphosphates in combination with Vent (exo-) DNA polymerase. DNAs modified by pairs of dissimilar nucleotides (dU + dC, dU + dA, dC + dA) with similar and different functional substituents were obtained.
Asunto(s)
ADN Polimerasa Dirigida por ADN , Nucleótidos , ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Purinas , PirimidinasRESUMEN
A prototype of a system for the detection of infectious human pneumonia pathogens based on multiplex solid-phase reverse transcription PCR (RT-PCR) was developed. Primers were designed to identify the DNA of six bacterial pneumonia pathogen strains, and the RNA of two viral pathogens of pneumonia: influenza A and SARS-CoV-2. The signal accumulation of elongated immobilized primers occurs due to the incorporation of fluorescently labeled nucleotides in the chain. The signal is detected after all the components of the mixture are removed, which significantly reduces the background signal and increases the sensitivity of the analysis. The use of a specialized detector makes it possible to read the signals of elongated primers directly through the transparent cover film of the reaction chamber. This solution is designed to prevent cross-contamination and is suitable for simultaneous testing of a large number of test samples. The proposed platform is able to detect the presence of several pathogens of pneumonia in a sample and has an open architecture that allows expansion of the range of pathogenic bacteria and viruses that can be detected.
RESUMEN
A prototype of a system for the detection of infectious human pneumonia pathogens based on multiplex solid-phase reverse transcription PCR (RT-PCR) was developed. Primers were designed to identify the DNA of six bacterial pneumonia pathogen strains, and the RNA of two viral pathogens of pneumonia: influenza A and SARS-CoV-2. The signal accumulation of elongated immobilized primers occurs due to the incorporation of fluorescently labeled nucleotides in the chain. The signal is detected after all the components of the mixture are removed, which significantly reduces the background signal and increases the sensitivity of the analysis. The use of a specialized detector makes it possible to read the signals of elongated primers directly through the transparent cover film of the reaction chamber. This solution is designed to prevent cross-contamination and is suitable for simultaneous testing of a large number of test samples. The proposed platform is able to detect the presence of several pathogens of pneumonia in a sample and has an open architecture that allows expansion of the range of pathogenic bacteria and viruses that can be detected.
Asunto(s)
COVID-19 , Transcripción Reversa , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
A multiplex PCR system has been developed and optimized for rapid detection of the five main pathogens of bacterial pneumonia. The system can be expanded to analyze viral pathogens of pneumonia (DNA- and RNA-containing viruses), as well as those of a fungal nature.
RESUMEN
Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.
Asunto(s)
Aptámeros de Nucleótidos/química , ADN/química , Biblioteca de Genes , Técnica SELEX de Producción de Aptámeros , Reacción en Cadena de la PolimerasaRESUMEN
Acral melanoma is one of the most aggressive and fast-growing forms of cutaneous melanoma and is characterized by a predominant location on the palms and feet. Primary tumors, metastases, and normal tissue samples from five acral melanoma patients were examined by massive parallel sequencing, focusing on the coding regions of 4100 genes involved in the origin and progression of hereditary and oncology diseases. Somatic mutations were found in genes related to cell division, proliferation, and apoptosis (BRAF, NRAS, VAV1, GATA1, and GCM2); cell adhesion (CTNND2 and ITGB4); angiogenesis (VEGFA); and the regulation of energy metabolism (BCS1L). Comparisons of target DNA sequences between morphologically normal and primary tumor tissues and between normal and metastatic tissues identified the candidate genes responsible for rapid metastasis in acral melanoma.
Asunto(s)
Melanoma/genética , Melanoma/patología , Mutación , Metástasis de la Neoplasia/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Humanos , Análisis de Secuencia de ADNRESUMEN
The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , ADN/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Biblioteca de Genes , Cinética , Técnica SELEX de Producción de AptámerosRESUMEN
The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo-) and Deep Vent (exo-) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chro-mophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.
Asunto(s)
Carbocianinas/química , ADN Polimerasa Dirigida por ADN/química , ADN/síntesis química , Nucleótidos de Desoxicitosina/química , ADN/química , Coloración y EtiquetadoRESUMEN
A biochip, primer set, and genotyping protocol were developed to simultaneously address 16 single nucleotide polymorphisms in antileukemic drug metabolism genes, including TPMT, ITPA, MTHFR, SLCO1B1, SLC19A1, NR3C1, GRIA1, ASNS, MTRR, and ABCB1. The genotyping procedure included a one-round multiplex polymerase chain reaction (PCR) with simultaneous incorporation of a fluorescent label into the PCR product and subsequent hybridization on a biochip with immobilized probes. The method was used to test 65 DNA samples of leukemia patients. Fluorescence signal intensity ratios in pairs of wild-type and respective mutant sequence probes were analyzed for all polymorphic markers and demonstrated high accuracy of genotyping. The reliability of genotype determination using the biochip was confirmed by direct Sanger sequencing.
Asunto(s)
Alelos , Antineoplásicos/farmacocinética , Técnicas de Genotipaje , Leucemia , Reacción en Cadena de la Polimerasa Multiplex , Proteínas de Neoplasias , Polimorfismo de Nucleótido Simple , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucemia/metabolismo , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
Biological microarrays (biochips) are analytical tools that can be used to implement complex integrative genomic and proteomic approaches to the solution of problems of personalized medicine (e.g., patient examination in order to reveal the disease long before the manifestation of clinical symptoms, assess the severity of pathological or infectious processes, and choose a rational treatment). The efficiency of biochips is predicated on their ability to perform multiple parallel specific reactions and to allow one to study the interactions of biopolymer molecules, such as DNA, proteins, glycans, etc. One of the pioneers of microarray technology was the Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences (EIMB), with its suggestion to immobilize molecular probes in the three-dimensional structure of a hydrophilic gel. Since the first experiments on sequencing by hybridization on oligonucleotide microarrays conducted some 30 years ago, the hydrogel microarrays designed at the EIMB have come a long and successful way from basic research to clinical laboratory diagnostics. This review discusses the key aspects of hydrogel microarray technology and a number of state-ofthe-art approaches for a multiplex analysis of DNA and the protein biomarkers of socially significant diseases, including the molecular genetic, immunological, and epidemiological aspects of pathogenesis.
RESUMEN
This study investigated the synthesis and substrate properties of Cy5-labeled dUTP derivatives with different substituents, linkers between the dye unit and pyrimidine heterocycle and fluorophore charges. Fluorescently labeled nucleoside triphosphates were studied as substrates using multiplex PCR with Taq and Vent (exo-) DNA polymerases, the typical representatives of the A and B polymerase families. The efficiency of nucleotide incorporation during PCR was assessed with a multi-parameter hybridization analysis using a diagnostic DNA microarray. The hybridization analysis indirectly estimates the incorporation efficiency of dye-labeled nucleotides in multiplex PCR. Our results demonstrated higher efficiencies of substrates with electrically neutral dyes than electropositive and electronegative Cy5 residues.
Asunto(s)
Carbocianinas/química , ADN/análisis , ADN/química , Colorantes Fluorescentes/química , Análisis por Micromatrices/métodos , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , ADN Polimerasa Dirigida por ADN/metabolismo , HumanosRESUMEN
Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN/biosíntesis , Técnica SELEX de Producción de Aptámeros , ADN/química , ADN/genética , Cartilla de ADN , ADN Polimerasa Dirigida por ADN/genética , Nucleótidos/síntesis química , Nucleótidos/química , Nucleótidos/genética , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Oligonucleótidos/genéticaRESUMEN
MLL is involved in fusion genes with more than 100 partner genes, approximately 80 of which have been characterized at the molecular level. MLL fusion genes are often found in infants (60-80% of acute lymphoblastic leukemia (ALL) cases and 40-50% of acute myeloblastic leukemia (AML) cases) and are appreciably rarer (8-10%) in children older than 1 year of age. MLL rearrangements are important markers in diagnosis and treatment choice. To identify the partner gene is of primary importance for prognosis and minimal residual disease monitoring. The structure of the fusion gene, including localization of the MLL breakpoints, is also informative. A method was developed to examine the fusion transcripts in order to identify the partner gene among the six most common ones and to establish the exon structure of the rearranged MLL. The method includes a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify and to fluorescently label a fusion transcript fragment and subsequent hybridization of the product on a biological microchip with immobilized oligonucleotides complementary to exons of MLL and its partner genes AFF1, MLLT1, MLLT3, MLLT4, MLLT10, and ELL. Hybridization results were verified by sequencing the RT-PCR products and, in some cases, performing long-distance inverse PCR (LDI-PCR). The study involved 38 bone marrow samples from ALL patients (including 33 children younger than 1 year of age) and 15 samples from AML patients (including 10 from children younger than 1 year of age). The main partner genes were AFF1 (49%), MLLT1 (27%), MLLT3 (12%), and MLLT10 (12%) in ALL and MLLT3 (80%), MLLT10 (10%), and MLLT4 (10%) in AML. Fusion gene transcripts most commonly included MLL exon 11 (58% of ALL cases and 50% of AML cases), suggesting a breakpoint in MLL intron 11.
Asunto(s)
Perfilación de la Expresión Génica/instrumentación , Regulación Leucémica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/biosíntesis , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Proteínas de Fusión Oncogénica/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transcripción Genética , Niño , Preescolar , Femenino , Perfilación de la Expresión Génica/métodos , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Lactante , Recién Nacido , Leucemia Mieloide Aguda/genética , Masculino , Proteína de la Leucemia Mieloide-Linfoide/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
Antipsychotics are the main drugs for the treatment of severe mental illness--schizophrenia affects about 1% of the population. The mechanism of action of neuroleptics is still up to the end. Several studies in the field of pharmacogenetics confirm enourmous influence of several neurotransmitter systems in the brain on the efficiency and the development of side effects. In this paper, we analyzed the association of nine polymorphic variants of five genes of dopaminergic and serotonergic systems DRD4, HTR2A, TPH1, SLC18A1, COMT in Russian and Tatars patients living in the Republic of Bashkortostan (RB) with the efficiency of a typical antipsychotic haloperidol on the scale of positive and negative systems of PANSS. The study established pharmacogenetic markers of increased and decreased effectiveness of therapy with haloperidol in the treatment groups. The results of this study confirm the importance of changes in the nucleotide sequences of the studied genes of the serotoninergic and dopaminergic systems (HTR2A, TPH1, SLC18A1 COMT, DRD4) in the formation of individual sensitivity to haloperidol. The results of our work considered as preliminary contact, requires an increase in the number of samples studied.
Asunto(s)
Antipsicóticos/farmacología , Haloperidol/farmacología , Polimorfismo de Nucleótido Simple , Esquizofrenia/genética , Transmisión Sináptica/genética , Adolescente , Adulto , Antipsicóticos/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Estudios de Casos y Controles , Catecol O-Metiltransferasa/genética , Femenino , Haloperidol/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Receptor de Serotonina 5-HT2A/genética , Receptores de Dopamina D4/genética , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/etnología , Tatarstán , Triptófano Hidroxilasa/genética , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Carbocianinas/síntesis química , Colorantes Fluorescentes/síntesis química , Análisis por Micromatrices/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carbocianinas/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Cartilla de ADN/síntesis química , Cartilla de ADN/genética , Femenino , Colorantes Fluorescentes/metabolismo , Expresión Génica , Humanos , Rayos Infrarrojos , Dispositivos Laboratorio en un Chip , Análisis por Micromatrices/instrumentación , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Reacción en Cadena de la Polimerasa/métodosAsunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Inhibidores Enzimáticos/farmacología , Virus de la Influenza A/genética , Análisis por Micromatrices/métodos , Adamantano/farmacología , Diseño de Equipo , Virus de la Influenza A/efectos de los fármacos , Análisis por Micromatrices/instrumentación , Mutación , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Proteínas de la Matriz Viral/genética , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genéticaRESUMEN
The aim of this work was to compare different speckle reduction techniques. It was shown that the use of devices based on liquid crystals only leads to partial reduction of speckle contrast. In quantitative luminescent microscopy an application of the mechanical devices when a laser beam is spread within the field of view turned out to be more efficient. Laser speckle noise was virtually eliminated with the developed and manufactured mechanical device comprising a fiber optic ring light guide and the vibrator that permits movement of optical fiber ends towards the laser diode during measurements. The method developed for the analysis of microarrays was successfully applied to the problem of speckle reduction.
Asunto(s)
Biofisica , Rayos Láser , Luminiscencia , Microscopía/métodos , Luz , Óptica y FotónicaRESUMEN
In order to study the effect of an electrical charge of the chromophore, on the efficiency of incorporation of fluorescently-labeled nucleotides into DNA during PCR, three fluorescently-labeled dUPT, one of which with electroneutral and other two with positively and negatively charged dyes (Cy5 analogs), were synthesized. It is shown that dUPT, labeled with electroneutral Cy 5 analog, is most effectively incorporated into DNA when Tag polymerase is used for PCR.