ABSTRACT
OBJECTIVES@#To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism (cSNP) based on SNaPshot technology in semen stains and mixed body fluid identification.@*METHODS@#Genomic DNA (gDNA) and total RNA were extracted from 16 semen stains and 11 mixtures composed of semen and venous blood, and the total RNA was reverse transcribed into complementary DNA (cDNA). The cSNP genetic markers were screened on the validated semen-specific mRNA coding genes. The cSNP multiplex detection system based on SNaPshot technology was established, and samples were genotyped by capillary electrophoresis (CE).@*RESULTS@#A multiplex detection system containing 5 semen-specific cSNPs was successfully established. In 16 semen samples, except the cSNP located in the TGM4 gene showed allele loss in cDNA detection results, the gDNA and cDNA typing results of other cSNPs were highly consistent. When detecting semen-venous blood mixtures, the results of cSNP typing detected were consistent with the genotype of semen donor and were not interfered by the genotype of venous blood donor.@*CONCLUSIONS@#The method of semen-specific cSNPs detection by SNaPshot technology method can be applied to the genotyping of semen (stains) and provide information for determining the origin of semen in mixed body fluids (stains).
Subject(s)
Genetic Markers , Semen , Polymorphism, Single Nucleotide , DNA, Complementary/genetics , Body Fluids , RNA, Messenger/genetics , DNA , Saliva , Forensic Genetics/methodsABSTRACT
@#Abstract: - （ ） ， Noise induced hearing loss NIHL is a hearing disorder that seriously harms the health of workers and it is a （ ） complex disease caused by environmental and genetic factors. Single nucleotide polymorphism SNP is the most common ， （ ） genetic variation which is associated with the occurrence and development of NIHL. MicroRNA miRNA is a kind of small - - ， - non codingsingle strandedRNA whichishighlyexpressedinthecochleaandregulatesgenesbybindingtothe3′ untranslated （ ） - regionoftargetmessengerRNA mRNA .SNPofmiRNA isthemostcommongeneticvariation.SNPinthenon codingregion， participates in gene expression regulation and phenotype generation by leading to abnormal miRNA recruitment thereby， affecting the occurrence and development of NIHL. The genetic susceptibility genes of NIHL include oxidative stress genes - singlegenedeafnessgenesandheatshockproteingenes.TheSNPinthenon codingregionisassociatedwiththesusceptibility， of NIHL. miRNA SNP has been confirmed to be involved in the development of NIHL but the correlation between different miRNASNPandNIHLisdifferent.SNPaffectsthesusceptibilityofNIHLbyaffectingtheexpressionandfunctionofmiRNA.
ABSTRACT
With the continuous development of bioinformatics technology and precision medicine, genetic mechanism investigations of genetic diseases including cleft lip and palate (CLP) have been getting more and more attention. Researchers have focused on the coding sequence of the genome and successfully found many CLP causative mutations, but there still remain some unsolved questions. In recent years, researchers′ vision has gradually shifted to non-protein coding region of the genome. This article reviews several coding sequence mutations, non-protein coding variants and their genetic mechanisms discovered in CLP researches.
ABSTRACT
Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5′-noncoding region (5′-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5′-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5′-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5′-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the HCV 5′-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
ABSTRACT
OBJECTIVE@#This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA.@*METHODS@#Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed.@*RESULTS@#The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation.@*CONCLUSIONS@#The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
Subject(s)
Humans , Computational Biology , DNA Methylation , DNA, Circular/genetics , DNA, Viral/genetics , Genome, Human , Genomics , Genotype , Hepacivirus/genetics , Hepatitis C/virology , Leukocytes, Mononuclear/virology , Polymerase Chain Reaction , RNA, Viral/genetics , Sequence AlignmentABSTRACT
Objective@#To analyze the phylogenetic characteristics of enterovirus 71 (EV71) in Xinjiang Production and Construction Corps from 2011 to 2016, providing pathogenic information for prevention and control of hand-foot-mouth disease (HFMD).@*Methods@#The EV71 positive strains were conducted for reverse transcription polymerase chain reactions (RT-PCR) amplification on entire VP1 coding region and sequencing was then performed and finally phylogcnetic tree was constructed among these strains and representative strains from GenBank using MEGA6.06.@*Results@#The homology of nucleotide and amino acid of the 37 EV71 strains were 87.3%-100.0% and 93.1%-100.0%, and belonged to EV71 C4a subgenotype, of 7 relatively independent small branches. Of the epidemic strains, compared with the representative strains, mutation occurred on 28 amino acid sites, the variation (Serine to Threonine, Alanine to Serine) happened on the 283 and 293 amino acid site in 9 strains.@*Conclusions@#The 37 EV71 strains in Xinjiang Production and Construction Corps from 2011 to 2016 belonged to EV71 C4a subgenotype, mutation occurred on 28 amino acid sites, and there is a common variation on the 283 and 293 amino acid site in 9 strains.
ABSTRACT
Objective To clone the coding region and 3′non?coding region of calmodulin 2(CaM2)in guinea pig,to provide the genetic informa?tion for studying the gene function of Calmodulin 2. Methods Total RNA was extracted from heart tissue of guinea pig,the coding region and 3′non?coding region of CaM2 were amplified by RT?PCR and 3′?RACE PCR methods,and the recombinant plasmid was constructed by inserting cDNA of the coding region and 3′non?coding region of CaM2 into the cloning vector by genetic engineering technology followed by DNA sequencing and se?quence analysis. Results The cloned coding region of CaM2 was 450 bp,and the 3′non?coding region of CaM2 was 660 bp. The amino acid se?quences of the coding region of CaM2 was consistent with those of other CaM subtypes,and the 3′non?coding region of CaM2 had low homology with those of other subtypes. Conclusion The cloning of CaM2 coding region and 3′non?coding region in guinea pig was the foundation for further study on the gene function of CaM2 and its role in related diseases.
ABSTRACT
Coding region instability determinant (CRD) is one of the influence factors of oncogene c-myc.Coding region instability determinant-binding protein (CRD-BP) can connect with CRD in order to protect CRD from nuclease attack,prevent rapid degradation of c-myc mRNA,and increase c-myc protein content.Beta-transducin repeats-containing protein (β-TrCP) can affect cell growth,differentiation,apoptosis and oncogenesis by regulating multiple signaling pathways and cell cycle.The overexpression of CRD-BP can upregulate the expression of β-TrCP and both of them play important roles in the tumorigenesis,progression,metastasis and invasion of colorectal cancer.
ABSTRACT
The primary clue for locating protein-coding regions is the open reading frame and the determination of ORFs (Open Reading Frames) is the first step toward the gene prediction, especially for prokaryotes. In this respect, we have developed a web-based ORF search tool called ORF Miner. The ORF Miner is a graphical analysis utility which determines all possible open reading frames of a selectable minimum size in an input sequence. This tool identifies all open reading frames using alternative genetic codes as well as the standard one and reports a list of ORFs with corresponding deduced amino acid sequences. The ORF Miner can be employed for sequence annotation and give a crucial clue to determination of actual protein-coding regions.
Subject(s)
Animals , Amino Acid Sequence , Ecthyma, Contagious , Genetic Code , Open Reading Frames , Resin CementsABSTRACT
As characterization of mitochondrial DNA (mtDNA) shows maternal inheritance and exists as more than thousands copies per cell, it is widely used for population genetics and forensic scientific field. However, mitochondrial DNA study has difficulties because heteroplasmy of mtDNA is being reported from coding and control region. In this study, we have analyzed 200 samples to examine heteroplasmy in mitochondrial DNA of Korean and Mongolian. The control region and coding region in mtDNA of blood from Koreans and Mongolians were analyzed with PCR amplication and sequencing. As a result, several heteroplasmy was observed from total 10 positions including 5 positions in coding region and 5 positions in control region, respectively. Moreover, it showed more than one heteroplasmy in coding region from 6 samples in Korean and 17 samples in Mongolian. Interestingly, heteroplasmy at 5178 position was shown in 6 samples among 23 samples. Considering that the position is important for deciding haplogroup D, we suggest that additional analysis on 4883 position needs for correct haplogrouping. Beside, we also found heteroplasmy in the other positions of 204, 4853, or 16249. Therefore, we suggest that it is required of combinatory analysis on several key nucleotide positions to obtain good results when determining mitochondrial haplogroups.
Subject(s)
Clinical Coding , Coat Protein Complex I , DNA, Mitochondrial , Genetics, Population , Polymerase Chain Reaction , WillsABSTRACT
Even though mitochondrial DNA analysis is performed in the field of molecular genetics, differences of the results exist regarding which nucleotide positions are analyzed. In this study, we strategically analyzed to find ethnic specific SNP of coding regions of mitochondrial DNA of Korean and Mongolian. Mitochondrial DNA was analyzed with PCR amplification and sequencing with 112 blood samples of Korean and 92 blood samples of Mongolian. As a result, the mutation which commonly appears both in Korean and Mongolian population is 17 nucleotide positions, and the one that shown in the only Korean is 13 nucleotide positions, the one that shown in the only Mongolian 26 nucleotide positions. However, it was thought as individual variation as most mutations are shown in a sample. Among them, it appears as 9% substitution rate in 10397, 4850 nucleotide position of Korean, whereas 12.3% or 15% substitution rate in 5108, 9950 nucleotide positions of Mongolian, respectively. Beside, we observed high level of heteroplasmy in 3546, 3553 nucleotide positions. Therefore, we suggest that these regions might be novel genetic markers for dividing mitochondrial haplogroup of Korean and Mongolian population, but additional analysis needs on several nucleotide positions in huge samples as analyzing on restricted nucleotide positions using restricted DNA samples.
Subject(s)
Clinical Coding , DNA , DNA, Mitochondrial , Genetic Markers , Molecular Biology , Polymerase Chain ReactionABSTRACT
Torque teno virus (TTV) is a recently discovered DNA virus that was originally isolated from a Japanese patient (initials, TT) with post-transfusion hepatitis of unknown aetiology. TTV is an circular DNA virus classified recently together with related Torque teño minivirus, into a new genus called Anellovirus. Infection TTV has been detected in a range of non-human primates as well as domestic animals. The purpose of this study was to search TTV in the serum and total blood of Brazilian monkeys and in plasma of domestic chickens by seminested PCR of coding region (N22), followed by a genomic sequence and phylogenetic analysis. No serum sample was amplified. TTV DNA was detected in total blood from 3 (4 percent) out of 75 brown-capuchin (Cebus apella) and from 1 (25 percent) out of 4 golden-headed lion-tamarin (Leontopithecus chrysomelas). Phylogenetic analysis revealed that one sample showed similarity with one sequence of the cotton top tamarin (Saguinus oedipus) (So-TTV2) and with one of the douroucoulis (ão tes trivirgatus) (At-TTV3). Two samples showed similarity with a human Torque Teño Mini Virus (TLMV). The other sample clustered with one sequence of the chimpanzee (Pt-TTV6) and with the human TTV strain TA278. The plasma chicken samples tested were all negative. The amino acid sequences reported in this study are the first obtained in Brazil from total blood of non-human primates naturally infected by TTV.
Torque teno virus (TTV) es un virus de ADN recientemente descubierto que fue inicialmente aislado de un paciente japonés (iniciales TT) después de la transfusión de hepatitis de etiología desconocida. TTV es un virus de ADN circular recientemente clasificado junto con los torque teno minivirus, en un nuevo género llamado Anellovirus. La infección de TTV se ha detectado en una serie de primates no humanos, así como animales domésticos. El objetivo de este estudio fue buscar TTV en el suero y sangre total de monos de Brasil y en el plasma de pollos domésticos, por seminested PCR de la región de codificación (N22), seguido de una secuencia genómica y el análisis filogenético. Las muestras que no eran suero fueron amplificadas. TTV DNA se detectó en sangre total de 3 (4 por ciento) de un total de 75 capuchinos de cabeza dura (Cebus apella) y de 1 (25 por ciento) de un total de 4 tití- león de cabeza dorada (Leontopithecus chrysomelas). El análisis filogenético demostró que una muestra presentaba similitud con una secuencia de Saguinus Edipo (So-TTV2) y con una de Aotes trivirgatus (A-TTV3). Dos muestras mostraron similitud con un torque teno mini virus (TLMV) humano. La otra muestra agrupada con una secuencia de los chimpancés (PT-TTV6) y con el TTV humanos cepa TA278. El análisis de las muestras de plasma de pollo fueron negativas Las secuencias de aminoácidos que se reportan en este estudio son las primeras obtenidas en Brasil de sangre de primates no humanos infectados naturalmente por TTV.
Subject(s)
Poultry Diseases/virology , Primate Diseases/virology , DNA Virus Infections/genetics , DNA Virus Infections/blood , DNA Virus Infections/veterinary , Torque teno virus/isolation & purification , DNA, Viral/genetics , DNA, Viral/blood , Amino Acid Sequence , Brazil , Poultry Diseases/genetics , Poultry Diseases/blood , Primate Diseases/genetics , Primate Diseases/blood , Genome, Viral , Phylogeny , Polymerase Chain Reaction , Chickens/virology , Primates/virologyABSTRACT
For evaluation of the five coding region (CR) polymorphism in mitochondrial DNA (mtDNA); we had performed PCR and direct sequencing in 599 unrelated Korean who showed the identical DNA type in D-loop mitochondrial DNA analysis for total 2,810 bp fragment. Following the sequence analysis, all the sequences of five regions were compared respectively to Anderson standard sequence to investigate the nucleotide variations. The result showed, a total 4,565 nucleotide variations were observed at 190 positions in five CR as 3,931 (86.11%) substitutions, 32 (0.7%) insertions, and 602 (13.19%) deletions and the allele diversities (h) were higher than 0.9992 when adding each CR or combined CR to D-loop analysis in mtDNA. In conclusion, we could confirm the five CR are useful for forensic testing through the nucleotide variation and hapolotypes polymorphism.
Subject(s)
Alleles , Clinical Coding , DNA , DNA, Mitochondrial , Polymerase Chain Reaction , Sequence AnalysisABSTRACT
The coding region of S genome segment of Hantaan virus (76/118 strain) was inserted into the eukarytic expression plasmidpVR1012. The recombinant expression plasmid pVRS22 was constructed. Vero-E6 cells were transiently transfected in vitro with pVRS22 plasmid. The transient expression of Hantaan virus nucleocapsid proteins in Vero-E6 cells was detected by indirect immunofluorescence assay (IFA) with monoclonal antibody 5H5 against Hantaan virus.