New lines of wheat ( Triticum aestivum L.) was obtained by introducing the DNA of sorghum (Sorghum vulgare Pers.) into wheat cultivar 'Longchun 13'. The changes of respiratory pathway, contents of protein, Na+...New lines of wheat ( Triticum aestivum L.) was obtained by introducing the DNA of sorghum (Sorghum vulgare Pers.) into wheat cultivar 'Longchun 13'. The changes of respiratory pathway, contents of protein, Na+ and K+ in the leaves and roots of the new lines of wheat under salt stress were determined and compared with the control cultivar, 'Longchun 13'. The decrease of the content of K+ was observed with the increase of NaCl concentrations, but the decrease was more in the control than that in the new lines, and more in roots than in leaves. Content of proline and Na+ in both two wheats lines increased greatly, but the former increased more significantly in the new lines and the latter more significantly in control both in leaves and roots. The operation of the cyanide-resistant pathway of respiration was enhanced at different degrees after salt stress and it increased much more in roots and leaves of the control plant than that in the new lines, but the cytochrome pathway of electron transport was still the main one consistently. The possible significance of these changes was discussed.展开更多
Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play ...Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play a critical role in the stabilization and establishment of nascent plant polyploids as new species. To further study this issue, five wheat chromosome 7B_specific sequences, isolated from common wheat (Triticum aestivum L.) by chromosome microdissection, were characterized. The sequences were studied by genomic Southern hybridizations on a collection of polyploid wheats and their diploid progenitors. Four sequences hybridized to all polyploid species, but at the diploid level to only species closely related to the B_genome of polyploid wheat. This indicates that these sequences originated with the divergence of the diploid species, and was then vertically transmitted to polyploids. One sequence hybridized to all species at both the diploid and polyploid levels, suggesting its elimination after the polyploid wheat formation. The hybridization of this sequence to two synthetic polyploid wheats indicated that sequence elimination is a rapid event and probably related to methylation status of the sequence. Based on the above results, we suggest that selective changes of low_copy sequences occur rapidly after polyploid formation, which may contribute to the differentiation of chromosomes in newly formed allopolyploid wheats.展开更多
With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measu...With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measurements in comparison with the binding between DNA and Hoechst 33258. The inhibition mode by YR and GGH to DNA binding of Hoechst 33258 was analyzed by Lineweaver-Burk plot which shows the plot of typical competitive inhibition at concentration of Hoechst 33258 from 3.66 ( 10-9 mol / L to 1.09 ( 10-8 mol / L. And it is concluded that YR binds to DNA in its minor groove (AT rich regions) with a binding constant K = 1.02 ( 108 (mol / L)-1. The GGH(s specificity is reduced at high concentration because it can also bind GC base pair.展开更多
DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the...DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.展开更多
Using the complete genome of Plasmodium falciparum 3D7 which has 14 chromosomes as an example, we have examined the distribution functions for the amount of C or G and A or T consecutively and non-overlapping blocks o...Using the complete genome of Plasmodium falciparum 3D7 which has 14 chromosomes as an example, we have examined the distribution functions for the amount of C or G and A or T consecutively and non-overlapping blocks of m bases in this system. The function P(S) about the number of the consecutive C-G or A-T content cluster conforms to the relation P(S)∝e? ; αs values of the scaling exponent αCG are much larger than αAT; and αAT of 14 chromosomes are hardly changed, whereas αCG of 14 chromosomes have a number of fluctuations. We found maximum value of A-T cluster size is much larger than C-G, which implies the existence of large A-T cluster. Our study of the width function ξ(m) of cluster C-G content showed that follows good power law ξ(m)∝m?γ. The average γ for 14 chromosomes is 0.931. These investigations provide some insight into the nucleotide clusters of DNA sequences, and help us understand other properties of DNA sequences.展开更多
We propose an Ashkin-Teller-like model for elastic response of DNA molecule to external force and torque. The base-stacking interaction is described in a simple and uniform way. We obtain the phase diagram of dsDNA, a...We propose an Ashkin-Teller-like model for elastic response of DNA molecule to external force and torque. The base-stacking interaction is described in a simple and uniform way. We obtain the phase diagram of dsDNA, and in particular, the transition from 13 form to the S state induced by stretching and twisting. The elastic response of the ssDNA is presented also in a unified formalism. The close relation of dsDNA molecule structure with elastic response is shown clearly. The calculated folding angle of the dsDNA molecule is 59.2°.展开更多
This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragment...This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.展开更多
Previous investigation on the mutagenic effects of 3,N4-Ethenocytosine (εC), a nonpairing DNA lesion,revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E. coli termed UVM for UV modula...Previous investigation on the mutagenic effects of 3,N4-Ethenocytosine (εC), a nonpairing DNA lesion,revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E. coli termed UVM for UV modulation of mutagenesis. To investigate whether UVM is mediated by an alteration of DNA replication, we have set up an in vitro replication system ill which phage M13 viral single-stranded DNA bearing a single site-specific (εC) residue is replicated by soluble protein extracts from E. coli cells. Replication products were analyzed by agarose gel electrophoresis and the frequency of translesion synthesis was determined by restriction endonuclease analyses. Our data indicate that DNA replication is strongly inhibited by εC, but that translesion DNA synthesis does occur in about 14% of the replicated DNA molecules. These results are very similar to those observed previously in vivo, and suggest that this experimental system may be suitable for evaluating alterations in DNA replication in UVM-induced cells.展开更多
Plant RNA N-glycosidase specifically hydrolyzes the N-C glycosidic bond of a conserved adenosine in the sarcin/ricin domain of the largest RNA in ribosome, releasing an adenine base and thus inhibiting protein synthes...Plant RNA N-glycosidase specifically hydrolyzes the N-C glycosidic bond of a conserved adenosine in the sarcin/ricin domain of the largest RNA in ribosome, releasing an adenine base and thus inhibiting protein synthesis. This substrate specificity was challenged later by discovery that various RNA derivatives and DNAs, especially the double-stranded supercoiled DNA could be used as substrate by RNA N-glycosidase. Thus, it was argued whether the DNA-cleaving activity was an intrinsic feature of RNA N-glycosidase or it was contaminated by DNase. In this article, several lines of evidence are presented to show that RNA N-glycosidase can really release the adenine base from the double-stranded supercoi/ed DNA. It was proposed that the cleavage mechanism of supercoiled DNA was the phosphodiester bonds in enzymatically deadenylated regions of the supercoiled DNA would become fragile and liable to produce nicked or linear form owing to the existence of tension in the supercoiled DNA molecule, not direct result of enzymatic action on the phosphodiester bond.展开更多
The origin and evolution of B chromosomes could be explained by the specific DNA sequence on them. But the specific sequences known were quite limited. To investigate maize B chromosome sqicific DNA sequeces, maize ge...The origin and evolution of B chromosomes could be explained by the specific DNA sequence on them. But the specific sequences known were quite limited. To investigate maize B chromosome sqicific DNA sequeces, maize genomes with and without B chromosomes were analyzed by AFLP. Only 5 markers were found specific to genomes with B chromosomes among about 2000 AFLP markers. Southern hybridization and sequence analysis revealed that only the sequence of M8-2D was a B chromosome specific sequence. This sequence contained the telomeric repeat unit AGGGTTT conserved in plant chromosome telomeres. In addition, the sequence of M8-2D shared low homology to clones from maize chromosome 4 centromere as well. M8-2D were localized to B chromosome centrorneric and telomeric regions.展开更多
Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellat...Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.展开更多
Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine...Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65℃ in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V, anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.展开更多
To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA (RAPD) was performed using 120 random primers. Of the suc...To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA (RAPD) was performed using 120 random primers. Of the successful amplicons obtained, the Panax ginseng-specific RAPD marker C-12 was cloned into a TA vector and sequenced (Genl3ank access number KU553472). Based on the sequence analysis results, a pair of primers specific to C-12 was designed. Finally, a SCAR marker-based identification system for Panax ginseng was developed after optimization of the reaction conditions. Using this method, two positive bands were stably observed at 300 bp and 130 bp in 33 batches of Panax ginseng samples tested, while negative results were obtained for another 101 batches of samples, including Panax quinquefolium, Panax notoginseng, adulterants, and other medicinal herbs. Thus, we successfully developed a PCR-based method for rapid and effective identification of Panax ginseng, which can be effectively used for the protection and utilization of germplasm resources and identification of the origins of Panax ginseng samples.展开更多
[Objective] This study established a method to detect Factor Ⅺ by polymerase chain reaction analysis.[Method]A pair of primers was designed and synthesized according to sequences of FⅪ gene in Holstein calves,publis...[Objective] This study established a method to detect Factor Ⅺ by polymerase chain reaction analysis.[Method]A pair of primers was designed and synthesized according to sequences of FⅪ gene in Holstein calves,published in Genbank. Polymerase chain reaction was used to analyze FⅪ deficiency of 576 Holstein calves in Henan,and the result was verified by DNA sequencing. [Result] We detect 576 cows,which include two carriers and one F Ⅺ deficiency,and the result was consistent with the DNA sequencing. The frequency of the FⅪ mutant allele was 0.3%,the carrier was 0.3%,the prevalence was 0.2%.[Conclusion]A method detecting FⅪ by polymerase chain reaction analysis was established. This method is not only simple and convenient,but also has a high accuracy and low cost,which is more suitable for large-scale FⅪ investigation.展开更多
Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus (HIV) infection diagnosis. Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 ...Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus (HIV) infection diagnosis. Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 was established based upon nicking-assisted strand displacement amplification (SDA) and triplex DNA. In the presence of target dsDNA, nicking-assisted SDA process generated a lot of ssDNA, which hybridized with molecular beacon to produce signal. The fluorescence intensity was proportional to the concentration of target dsDNA within the range from 5 to 1000 pmol/L, with a detection limit of 1.4 pmol/L. Moreover, it successfully distinguished target dsDNA from the nucleic acid extractive of human blood. Thus this method has the merit of high sensitivity, and it is suitable for sequence-specific recognition of target dsDNA in complex matrices, which made it a potential application in diagnosis of acquired immunodeflciency syndrome (AIDS) in the future.展开更多
Here, we report a method that uses gold nanoparticles (AuNPs) to enhance the specificity of DNA hybridization without reducing its detection sensitivity. The conventional stringent wash method utilizes high-temperat...Here, we report a method that uses gold nanoparticles (AuNPs) to enhance the specificity of DNA hybridization without reducing its detection sensitivity. The conventional stringent wash method utilizes high-temperatureflow-salt conditions to enhance the specificity of DNA hybridization-based assays. This method creates a destabilizing environment for base pairing that affects specific and nonspecific duplexes. Therefore, specificity is achieved at the expense of signal intensity or sensitivity. However, in the proposed wash method, AuNPs predominantly destabilize nonspecific duplexes, offering specificity without compromising sensitivity. This AuNP wash technique has proven to be effective in detecting single nucleotide polymorphisms (SNPs) in genomic samples even at room temperature in a CD-like NanoBioArray (CD-NBA) chip. This method is also robust with sequence variation and is compatible with multiplex DNA analyses on microarrays. Thus, the AuNP wash method could potentially be useful for improving the accuracy of DNA hybridization results.展开更多
文摘New lines of wheat ( Triticum aestivum L.) was obtained by introducing the DNA of sorghum (Sorghum vulgare Pers.) into wheat cultivar 'Longchun 13'. The changes of respiratory pathway, contents of protein, Na+ and K+ in the leaves and roots of the new lines of wheat under salt stress were determined and compared with the control cultivar, 'Longchun 13'. The decrease of the content of K+ was observed with the increase of NaCl concentrations, but the decrease was more in the control than that in the new lines, and more in roots than in leaves. Content of proline and Na+ in both two wheats lines increased greatly, but the former increased more significantly in the new lines and the latter more significantly in control both in leaves and roots. The operation of the cyanide-resistant pathway of respiration was enhanced at different degrees after salt stress and it increased much more in roots and leaves of the control plant than that in the new lines, but the cytochrome pathway of electron transport was still the main one consistently. The possible significance of these changes was discussed.
文摘Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play a critical role in the stabilization and establishment of nascent plant polyploids as new species. To further study this issue, five wheat chromosome 7B_specific sequences, isolated from common wheat (Triticum aestivum L.) by chromosome microdissection, were characterized. The sequences were studied by genomic Southern hybridizations on a collection of polyploid wheats and their diploid progenitors. Four sequences hybridized to all polyploid species, but at the diploid level to only species closely related to the B_genome of polyploid wheat. This indicates that these sequences originated with the divergence of the diploid species, and was then vertically transmitted to polyploids. One sequence hybridized to all species at both the diploid and polyploid levels, suggesting its elimination after the polyploid wheat formation. The hybridization of this sequence to two synthetic polyploid wheats indicated that sequence elimination is a rapid event and probably related to methylation status of the sequence. Based on the above results, we suggest that selective changes of low_copy sequences occur rapidly after polyploid formation, which may contribute to the differentiation of chromosomes in newly formed allopolyploid wheats.
文摘With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measurements in comparison with the binding between DNA and Hoechst 33258. The inhibition mode by YR and GGH to DNA binding of Hoechst 33258 was analyzed by Lineweaver-Burk plot which shows the plot of typical competitive inhibition at concentration of Hoechst 33258 from 3.66 ( 10-9 mol / L to 1.09 ( 10-8 mol / L. And it is concluded that YR binds to DNA in its minor groove (AT rich regions) with a binding constant K = 1.02 ( 108 (mol / L)-1. The GGH(s specificity is reduced at high concentration because it can also bind GC base pair.
文摘DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.
基金Project supported by the National Natural Science Foundation ofChina (Nos. 20174036 20274040)+2 种基金 and the Natural Science Founda-tion of Zhejiang Province (Nos. R404047 10102) China
文摘Using the complete genome of Plasmodium falciparum 3D7 which has 14 chromosomes as an example, we have examined the distribution functions for the amount of C or G and A or T consecutively and non-overlapping blocks of m bases in this system. The function P(S) about the number of the consecutive C-G or A-T content cluster conforms to the relation P(S)∝e? ; αs values of the scaling exponent αCG are much larger than αAT; and αAT of 14 chromosomes are hardly changed, whereas αCG of 14 chromosomes have a number of fluctuations. We found maximum value of A-T cluster size is much larger than C-G, which implies the existence of large A-T cluster. Our study of the width function ξ(m) of cluster C-G content showed that follows good power law ξ(m)∝m?γ. The average γ for 14 chromosomes is 0.931. These investigations provide some insight into the nucleotide clusters of DNA sequences, and help us understand other properties of DNA sequences.
基金National Natural Science Foundation of China under Grant Nos.10575106 and 10375072
文摘We propose an Ashkin-Teller-like model for elastic response of DNA molecule to external force and torque. The base-stacking interaction is described in a simple and uniform way. We obtain the phase diagram of dsDNA, and in particular, the transition from 13 form to the S state induced by stretching and twisting. The elastic response of the ssDNA is presented also in a unified formalism. The close relation of dsDNA molecule structure with elastic response is shown clearly. The calculated folding angle of the dsDNA molecule is 59.2°.
文摘This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among ge-nomic DNA. cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells. DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vec-tor respectively Fusion GST-Myc and GST-Myn synthe-sized in E.coli hosts showed affinity to CACGTG E-boxDNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR. A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA. At least two genomic DNA fragments ob- tained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone. Significance of the work and of the technique itself as well as identification of the DNAs are discussed.
文摘Previous investigation on the mutagenic effects of 3,N4-Ethenocytosine (εC), a nonpairing DNA lesion,revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E. coli termed UVM for UV modulation of mutagenesis. To investigate whether UVM is mediated by an alteration of DNA replication, we have set up an in vitro replication system ill which phage M13 viral single-stranded DNA bearing a single site-specific (εC) residue is replicated by soluble protein extracts from E. coli cells. Replication products were analyzed by agarose gel electrophoresis and the frequency of translesion synthesis was determined by restriction endonuclease analyses. Our data indicate that DNA replication is strongly inhibited by εC, but that translesion DNA synthesis does occur in about 14% of the replicated DNA molecules. These results are very similar to those observed previously in vivo, and suggest that this experimental system may be suitable for evaluating alterations in DNA replication in UVM-induced cells.
文摘Plant RNA N-glycosidase specifically hydrolyzes the N-C glycosidic bond of a conserved adenosine in the sarcin/ricin domain of the largest RNA in ribosome, releasing an adenine base and thus inhibiting protein synthesis. This substrate specificity was challenged later by discovery that various RNA derivatives and DNAs, especially the double-stranded supercoiled DNA could be used as substrate by RNA N-glycosidase. Thus, it was argued whether the DNA-cleaving activity was an intrinsic feature of RNA N-glycosidase or it was contaminated by DNase. In this article, several lines of evidence are presented to show that RNA N-glycosidase can really release the adenine base from the double-stranded supercoi/ed DNA. It was proposed that the cleavage mechanism of supercoiled DNA was the phosphodiester bonds in enzymatically deadenylated regions of the supercoiled DNA would become fragile and liable to produce nicked or linear form owing to the existence of tension in the supercoiled DNA molecule, not direct result of enzymatic action on the phosphodiester bond.
基金This research was supported by grant from National Natural Sciences Foundationof China (39970357).
文摘The origin and evolution of B chromosomes could be explained by the specific DNA sequence on them. But the specific sequences known were quite limited. To investigate maize B chromosome sqicific DNA sequeces, maize genomes with and without B chromosomes were analyzed by AFLP. Only 5 markers were found specific to genomes with B chromosomes among about 2000 AFLP markers. Southern hybridization and sequence analysis revealed that only the sequence of M8-2D was a B chromosome specific sequence. This sequence contained the telomeric repeat unit AGGGTTT conserved in plant chromosome telomeres. In addition, the sequence of M8-2D shared low homology to clones from maize chromosome 4 centromere as well. M8-2D were localized to B chromosome centrorneric and telomeric regions.
基金supported by the National Basic Research Program of China (973 Program) (No.2011CB403602)the National High Technology Research and Development Program of China (863 Program) (No.2007AA09200111)the National Marine Public Welfare Research Project (201205031-02)
文摘Prorocentrum donghaiense is a dinoflagellate that is widely distributed in the East China Sea and has become increasingly involved in Harmful Algal Blooms (HABs). Therefore, it is necessary to study this dinoflagellate to monitor HABs. In this study, 13 pairs of primers specific to P. donghaiense (within its internal transcribed spacer (ITS) regions) were designed for SYBR Green I real-time PCR. As the SYBR Green I real-time PCR could not identify P. donghaiense in a specific manner, a Taqman real-time PCR method was developed by designing a set of specific primers and a Taqman probe. A 10-fold serial dilution of recombinant plasmid containing ITS regions of P. donghaiense was prepared as standard samples and the standard curve was established. Additionally, we quantified the genomic DNA in P. donghaiense cells and utilized this DNA to prepare another 10-fold serial dilution of standard sample and accordingly set up the standard curve. The mathematic correlation between the cell number and its corresponding plasmid copy number was also established. In order to test the efficiency of the real-time PCR method, laboratory samples and P. donghaiense HAB field samples were employed for identification and quantitative analysis. As to laboratory samples, as few as 102 cells of P. donghaiense could be quantified precisely utilizing both centrifugation and filtration techniques. The quantification results from field samples by real-time PCR were highly similar to those by light microscopy. In conclusion, the real-time PCR could be applied to identify and quantify P. donghaiense in HABs.
基金Supported by the National Basic Research Program of China (973 Program) (No. 2006CB101804)the General Administration of Quality Supervision,Inspection and Quarantine of the People's Republic of China (No. 2007IK167)National Department Public Benefit Research Foundation (No. 200803012)
文摘Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65℃ in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V, anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.
基金Project(2014ZX09304307-002)supported by the Major Program of Science and Technology Foundation of ChinaProject supported by Technology Platform for Quality/Safety Inspection and Risk Management of Traditional Chinese Medicine,China+1 种基金Project(2014SK2001)supported by the Key Program Foundation of Hunan Provincial Science&Technology Department,ChinaProject(XSYK-R201502)supported by the Hunan Provincial Food and Drug Administration under Key Project of Science and Technology for Food and Drug Safety,China
文摘To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA (RAPD) was performed using 120 random primers. Of the successful amplicons obtained, the Panax ginseng-specific RAPD marker C-12 was cloned into a TA vector and sequenced (Genl3ank access number KU553472). Based on the sequence analysis results, a pair of primers specific to C-12 was designed. Finally, a SCAR marker-based identification system for Panax ginseng was developed after optimization of the reaction conditions. Using this method, two positive bands were stably observed at 300 bp and 130 bp in 33 batches of Panax ginseng samples tested, while negative results were obtained for another 101 batches of samples, including Panax quinquefolium, Panax notoginseng, adulterants, and other medicinal herbs. Thus, we successfully developed a PCR-based method for rapid and effective identification of Panax ginseng, which can be effectively used for the protection and utilization of germplasm resources and identification of the origins of Panax ginseng samples.
文摘[Objective] This study established a method to detect Factor Ⅺ by polymerase chain reaction analysis.[Method]A pair of primers was designed and synthesized according to sequences of FⅪ gene in Holstein calves,published in Genbank. Polymerase chain reaction was used to analyze FⅪ deficiency of 576 Holstein calves in Henan,and the result was verified by DNA sequencing. [Result] We detect 576 cows,which include two carriers and one F Ⅺ deficiency,and the result was consistent with the DNA sequencing. The frequency of the FⅪ mutant allele was 0.3%,the carrier was 0.3%,the prevalence was 0.2%.[Conclusion]A method detecting FⅪ by polymerase chain reaction analysis was established. This method is not only simple and convenient,but also has a high accuracy and low cost,which is more suitable for large-scale FⅪ investigation.
基金supported by the National Natural Science Foundation of China(21375153)
文摘Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus (HIV) infection diagnosis. Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 was established based upon nicking-assisted strand displacement amplification (SDA) and triplex DNA. In the presence of target dsDNA, nicking-assisted SDA process generated a lot of ssDNA, which hybridized with molecular beacon to produce signal. The fluorescence intensity was proportional to the concentration of target dsDNA within the range from 5 to 1000 pmol/L, with a detection limit of 1.4 pmol/L. Moreover, it successfully distinguished target dsDNA from the nucleic acid extractive of human blood. Thus this method has the merit of high sensitivity, and it is suitable for sequence-specific recognition of target dsDNA in complex matrices, which made it a potential application in diagnosis of acquired immunodeflciency syndrome (AIDS) in the future.
文摘Here, we report a method that uses gold nanoparticles (AuNPs) to enhance the specificity of DNA hybridization without reducing its detection sensitivity. The conventional stringent wash method utilizes high-temperatureflow-salt conditions to enhance the specificity of DNA hybridization-based assays. This method creates a destabilizing environment for base pairing that affects specific and nonspecific duplexes. Therefore, specificity is achieved at the expense of signal intensity or sensitivity. However, in the proposed wash method, AuNPs predominantly destabilize nonspecific duplexes, offering specificity without compromising sensitivity. This AuNP wash technique has proven to be effective in detecting single nucleotide polymorphisms (SNPs) in genomic samples even at room temperature in a CD-like NanoBioArray (CD-NBA) chip. This method is also robust with sequence variation and is compatible with multiplex DNA analyses on microarrays. Thus, the AuNP wash method could potentially be useful for improving the accuracy of DNA hybridization results.