At present,the polymerase chain reaction(PCR)amplification-based file retrieval method is the mostcommonly used and effective means of DNA file retrieval.The number of orthogonal primers limitsthe number of files that...At present,the polymerase chain reaction(PCR)amplification-based file retrieval method is the mostcommonly used and effective means of DNA file retrieval.The number of orthogonal primers limitsthe number of files that can be accurately accessed,which in turn affects the density in a single oligo poolof digital DNA storage.In this paper,a multi-mode DNA sequence design method based on PCR file retrie-val in a single oligonucleotide pool is proposed for high-capacity DNA data storage.Firstly,by analyzingthe maximum number of orthogonal primers at each predicted primer length,it was found that the rela-tionship between primer length and the maximum available primer number does not increase linearly,and the maximum number of orthogonal primers is on the order of 10^(4).Next,this paper analyzes themaximum address space capacity of DNA sequences with different types of primer binding sites for filemapping.In the case where the capacity of the primer library is R(where R is even),the number ofaddress spaces that can be mapped by the single-primer DNA sequence design scheme proposed in thispaper is four times that of the previous one,and the two-level primer DNA sequence design scheme can reach [R/2·(R/2-1)]^(2)times.Finally,a multi-mode DNA sequence generation method is designed based onthe number of files to be stored in the oligonucleotide pool,in order to meet the requirements of the ran-dom retrieval of target files in an oligonucleotide pool with large-scale file numbers.The performance ofthe primers generated by the orthogonal primer library generator proposed in this paper is verified,andthe average Gibbs free energy of the most stable heterodimer formed between the orthogonal primersproduced is−1 kcal·(mol·L^(−1))^(−1)(1 kcal=4.184 kJ).At the same time,by selectively PCR-amplifying theDNA sequences of the two-level primer binding sites for random access,the target sequence can be accu-rately read with a minimum of 10^(3) reads,when the primer binding site sequences at different positionsare mutually different.This paper provides a pipeline for orthogonal primer library generation and multi-mode mapping schemes between files and primers,which can help achieve precise random access to filesin large-scale DNA oligo pools.展开更多
Despite being ubiquitous in oceans and important in marine biogeochemical cycles,planktonic archaea in the Southern Ocean(SO)remain poorly characterized.Although high-throughput sequencing(HTS)approaches based on 16S ...Despite being ubiquitous in oceans and important in marine biogeochemical cycles,planktonic archaea in the Southern Ocean(SO)remain poorly characterized.Although high-throughput sequencing(HTS)approaches based on 16S ribosomal RNA(rRNA)genes have been used widely to study the diversity and composition of microbial community in natural environments,primer-set selection is critical because of amplicon-sequencing bias during metabarcoding.Here,using surface-seawater samples collected from the area between the South Shetland and South Orkney Islands,Antarctica,we compared primer sets Arch349F/Arch806R,515F-Y/926R,and 524F/Arch958R,which target different 16S rRNA gene hypervariable regions to identify the best one for studying planktonic archaeal communities.With much lower number of bacteria-related sequences,primer set 524F/Arch958R showed higher values of archaeal operational taxonomic units(OTUs)as well as alpha-diversity indices,indicating that this primer set was more specific for detecting archaeal species and could be helpful to obtain more comprehensive information on the archaeal community compositions compared to other two primer sets.Compared with primer set Arch349F/Arch806R revealing four phyla(Halobacteriota,Methanobacteriota,Thermoplasmatota,and Thermoproteota)detected in seawater,additional archaeal phyla were observed by 515F-Y/926R(Asgardarchaeota and Nanoarchaeota)and 524F/Arch958R(Micrarchaeota).In spite of the differences in archaeal community compositions observed among the three investigated primer sets,ammonia-oxidizing(e.g.,Nitrososphaeria)and methane-producing(e.g.,Methanobacteria,Methanomicrobia,and Methanosarcinia)archaea were the main groups detected in the surface seawater,indicating the ecological role of planktonic archaea in carbon and nitrogen cycling in the upper waters of the SO.These results underscore the importance of primer-set selection when studying archaeal community diversity and composition in the Antarctic SO.展开更多
[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 su...[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.展开更多
[Objective] This study aimed to screen a set of SSR core primers suitable for purity identification of pepper （Capsicum） hybrids. [Method] DNA fingerprint of 100 pepper hybrids was analyzed using 17 SSR primers. [Re...[Objective] This study aimed to screen a set of SSR core primers suitable for purity identification of pepper （Capsicum） hybrids. [Method] DNA fingerprint of 100 pepper hybrids was analyzed using 17 SSR primers. [Result] According to the polymorphism and heterozygosity, Hpms1-214, Es395 and Hpmsl-5 were determined as three preferred core primers for purity identification of pepper hybrids. By using these three preferred core primers, 97 pepper hybrids （accounting for 97%） had heterozygous band pattern with at least one primer. Es330, Es363, Epms923, Es120 and Es64 were determined as candidate core primers for purity identification of pepper hybrids. Specific primers of 14 varieties were obtained, which could be used to further screen parent-complementary primers of each pepper hybrid. [Con- clusion] This study laid the foundation for constructing standard DNA fingerprints for purity identification of pepper hybrids.展开更多
[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional tou...[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.展开更多
目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设...目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。展开更多
基金supported by the fund from Tianjin Municipal Science and Technology Bureau(22JCYBJC01390).
文摘At present,the polymerase chain reaction(PCR)amplification-based file retrieval method is the mostcommonly used and effective means of DNA file retrieval.The number of orthogonal primers limitsthe number of files that can be accurately accessed,which in turn affects the density in a single oligo poolof digital DNA storage.In this paper,a multi-mode DNA sequence design method based on PCR file retrie-val in a single oligonucleotide pool is proposed for high-capacity DNA data storage.Firstly,by analyzingthe maximum number of orthogonal primers at each predicted primer length,it was found that the rela-tionship between primer length and the maximum available primer number does not increase linearly,and the maximum number of orthogonal primers is on the order of 10^(4).Next,this paper analyzes themaximum address space capacity of DNA sequences with different types of primer binding sites for filemapping.In the case where the capacity of the primer library is R(where R is even),the number ofaddress spaces that can be mapped by the single-primer DNA sequence design scheme proposed in thispaper is four times that of the previous one,and the two-level primer DNA sequence design scheme can reach [R/2·(R/2-1)]^(2)times.Finally,a multi-mode DNA sequence generation method is designed based onthe number of files to be stored in the oligonucleotide pool,in order to meet the requirements of the ran-dom retrieval of target files in an oligonucleotide pool with large-scale file numbers.The performance ofthe primers generated by the orthogonal primer library generator proposed in this paper is verified,andthe average Gibbs free energy of the most stable heterodimer formed between the orthogonal primersproduced is−1 kcal·(mol·L^(−1))^(−1)(1 kcal=4.184 kJ).At the same time,by selectively PCR-amplifying theDNA sequences of the two-level primer binding sites for random access,the target sequence can be accu-rately read with a minimum of 10^(3) reads,when the primer binding site sequences at different positionsare mutually different.This paper provides a pipeline for orthogonal primer library generation and multi-mode mapping schemes between files and primers,which can help achieve precise random access to filesin large-scale DNA oligo pools.
基金The National Key Research and Development Program of China under contract No.2022YFC2807501.
文摘Despite being ubiquitous in oceans and important in marine biogeochemical cycles,planktonic archaea in the Southern Ocean(SO)remain poorly characterized.Although high-throughput sequencing(HTS)approaches based on 16S ribosomal RNA(rRNA)genes have been used widely to study the diversity and composition of microbial community in natural environments,primer-set selection is critical because of amplicon-sequencing bias during metabarcoding.Here,using surface-seawater samples collected from the area between the South Shetland and South Orkney Islands,Antarctica,we compared primer sets Arch349F/Arch806R,515F-Y/926R,and 524F/Arch958R,which target different 16S rRNA gene hypervariable regions to identify the best one for studying planktonic archaeal communities.With much lower number of bacteria-related sequences,primer set 524F/Arch958R showed higher values of archaeal operational taxonomic units(OTUs)as well as alpha-diversity indices,indicating that this primer set was more specific for detecting archaeal species and could be helpful to obtain more comprehensive information on the archaeal community compositions compared to other two primer sets.Compared with primer set Arch349F/Arch806R revealing four phyla(Halobacteriota,Methanobacteriota,Thermoplasmatota,and Thermoproteota)detected in seawater,additional archaeal phyla were observed by 515F-Y/926R(Asgardarchaeota and Nanoarchaeota)and 524F/Arch958R(Micrarchaeota).In spite of the differences in archaeal community compositions observed among the three investigated primer sets,ammonia-oxidizing(e.g.,Nitrososphaeria)and methane-producing(e.g.,Methanobacteria,Methanomicrobia,and Methanosarcinia)archaea were the main groups detected in the surface seawater,indicating the ecological role of planktonic archaea in carbon and nitrogen cycling in the upper waters of the SO.These results underscore the importance of primer-set selection when studying archaeal community diversity and composition in the Antarctic SO.
基金Supported by Important Project of Jinlin Provincial Science and Technology Department(20065020)~~
文摘[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.
基金Supported by Excellent Team Training Program of Yunnan Academy of Agriculture Sciences(YAAS2014YY002)~~
文摘[Objective] This study aimed to screen a set of SSR core primers suitable for purity identification of pepper （Capsicum） hybrids. [Method] DNA fingerprint of 100 pepper hybrids was analyzed using 17 SSR primers. [Result] According to the polymorphism and heterozygosity, Hpms1-214, Es395 and Hpmsl-5 were determined as three preferred core primers for purity identification of pepper hybrids. By using these three preferred core primers, 97 pepper hybrids （accounting for 97%） had heterozygous band pattern with at least one primer. Es330, Es363, Epms923, Es120 and Es64 were determined as candidate core primers for purity identification of pepper hybrids. Specific primers of 14 varieties were obtained, which could be used to further screen parent-complementary primers of each pepper hybrid. [Con- clusion] This study laid the foundation for constructing standard DNA fingerprints for purity identification of pepper hybrids.
文摘[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.
基金This work is supported by the National Natural Science Foundation of China(30070958).
文摘目的:通过使用3’末端碱基游移混合引物,减少引物末端错配,提高多变区基因片段的PCR检测阳性率。方法:在HBV DNA P基因区设计一对检测阳性率相对较高的引物(原型引物),在原型引物序列基础上将两根引物的3’末端分别截去1个或2个碱基,设计出两对参数与原型引物近似的突变引物。分别单独用原型引物和原型引物与突变引物组成的混合引物,在相同条件下对27份HBV DNA阳性标本行PCR,比较二者阳性率。用混合引物扩增4例拉米呋丁治疗无效患者血清HBV DNA,将扩增产物测序并评价3’末端错配对PCR的影响。结果:原型引物和混合引物检测阳性率分别为70.4％(19/27)和85.2％(23/27),两者差异非常显著(P<0.05)。引物3’末端错配能导致PCR假阴性。结论:扩增保守性相对较差的基因片段,采用3’末端单个或多个碱基截短的引物,构成3’末端碱基游移混合引物,可以避免因3’末端错配产生的PCR假阴性,提高检测阳性率。
