Amplification of cytochrome C oxidase summit I gene of Brandt's Vole by nested PCR.
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Abstract:
Objective
To determine a method for amplification of cytochrome C oxidase subunit I(CO I) gene of Brandt's vole.
Methods
The Brandt's Voles were captured in Abagaqi Xilingol League Inner Mongolia, and DNA was extracted from liver tissue. CO I gene was amplified by nested PCR and sequenced afterwards.
Results
A band of 657 bp and 1 132 bp was amplified by internal and external PCR primers, respectively, which were consistent with expected sizes. A total of 12 segments of Brandt's Vole CO I gene sequences were amplified by PCR and verified by sequencing. The sequence number was KF182196 - KF182207 in GenBank. After gene sequence alignment of the 12 CO I gene sequences, it was found that the similarity was 100%, and no base mutation.
Conclusion
CO I gene of Brandt's Vole could be amplified by nested PCR without pseudo gene.
Key words:
Nested PCR; Brandt's Vole; Cytochrome C oxidase subunit I geneKeywords:
Vole
AIM:To clone and analyse part of the sequence of 16S ribosomal RNA(16S rRNA) gene of Porphyromonas gingivalis(Pg ).METHODS:Polymerase chain reaction(PCR) was used to generate part of the 16S rRna gene of Pg ,then the gene was cloned and sequence analysed.RESULTS:A segment of DNA,size of which was 198 bp,was obtained and sequenced.The sequence was consistent with that displayed in Genbank on pubmed.CONCLUSION:Part of 16S rRNA gene was generated by PCR that could be the foundation of detection of Pg .
Cloning (programming)
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This study was based on a 278 and 635 bp region of the gene for cytochrome oxidase subunit I and I (CO I and CO II ) encoding region of mtDNA; the aim was to solve the problems in identifying Sarcosaphagous flies, particularly in the flies' larvae and eggs which could not be identified only by use of their morphologyical features.Samples of sarcosaphagous flies and larvae were collected from those on the corps of rabbits on the grassland in the Huhhot district and of a pig on the sandy ground in the Dunhuang district. The mtDNA of flies and their larvae and eggs was extracted using the Chelex technique. Polymerase chain reactions were conducted on a Perkin-Elmer 9600 thermal cycler, followed by vertical non-denaturing polyacrylamide gelectrophoresis. PCR products were purified using the Nucleic Acid Purification Kit. Sequences of both strands were obtained by direct sequencing of the double-stranded PCR product using one of the PCR primers and the ABI PRISM Big Dye Terminator Cycle Sequencing Kit. Sequence reactions were electrophoresed on ABI Model 377 DNA Sequencers. A neighbour-joining tree using the Tamura and Nei model of nucleotide substitution was constructed using the MEGA2. 1 package.A 278 and 635 bp region of the gene for CO I and CO II encoding region of mtDNA of Sarcosaphagous flies and their larvae and eggs was noted to show the percentages for the sequence divergence within species (less than 1%) and the sequence divergence between species (above 3%). For species that diverged from all others by a relatively large percentage and had small within-species variation, the least percentages of sequence divergence were given which distinguished any individual within that species from any other species.A 278 and 635 bp region of the gene for CO I and CO II encoding region of mtDNA of Sarcosaphagous flies and their larvae and eggs has been effectively used for the molecular phylogeny and the identification of their species group. CO I and CO II, or CO II, is better than CO I.
Chrysomya megacephala
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Mitochondrial 16S rRNA and COl gene fragments of F.chinensis were amplified via PCR,and then the PCR products were purified and sequenced.For 16S rRNA gene fragments,515 bp nucleotide sequences were obtained,and the A+T and G+C contents in this fragment were respectively 66.47%,33.53 %.As for the COI gene fragments,the size was 472 bp and the A+T and G+C contents were 62.50%,37.29% respectively.Analysis of 16S rRNA and COI gene fragments indicated that: 1) the AT content was higher than CG content,which was similar to the results of studies on drosophila,shrimp,crab and other invertebrates;2) four families from the test had 6 variable nucleotide positions of 16S rRNA gene fragments,but there were twenty-four variable nucleotide positions about the sequence of COI gene fragments.In addition,it is found that the graphical phylogenetic tree of 16S gene fragment of five shrimps in shrimp family is consistent with current systematic of these species.
GC-content
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To investigate the inter-specific genetic marker of Parabronema skrjabini,the genomic DNA of P.skrjabini collected from the infected camels in Inner Mongolia were extracted.The mitochondrial cytochrome oxidase subunit 1(CO1) gene was amplified by PCR using universal primers,and then the PCR product was cloned into pMD19-T vectors.The insert was sequenced successfully and compared with other nematode sequences by DNAStar 5.0 and MEGA 4.0.The results showed that the PCR product was 689 bp in length.Compared with other CO1 gene sequences of related nematode,the homology among P.skrjabini,Habronema microstoma,and H.muscae is 86.7% and the nucleotide pairwise distance is 0.143.Conversely,the homology between P.skrjabini and Litomosoides galizai is 80.1% and the nucleotide pairwise distance is 0.229.It suggested that the CO1 gene of P.skrjabini can be not only used as a genetic marker,but also used for further study in molecular classification.
Cloning (programming)
Homology
genomic DNA
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In this study,we designed primers according to sable gene and obtained the complete mitochon-drial 12S rRNA gene sequences of 7 breeds of mink by PCR amplification,sequencing and assembling.Sequence analysis revealed that 12S rRNA gene of minks was 960 or 962 bp in length,the composition of the nucleotides was 37.50%A,17.60%G,22.19%T and 22.71%C.8 variable sites were observed,and the transition/transversion(Ts/Tv)was 3.Given sable as outgroup and homologous sequences of 11 species in the mustela retrieved from GenBank,the phylogenetic trees were constructed respectively by N-J and MP methods.The results indicated that minks in China compared with American minks had closer relationship than that with European mink.
Transversion
Sequence (biology)
Maximum parsimony
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Mitochondrial 16S rRNA and COI gene fragments of Portunus trituberculatus from Weifang were amplified via PCR, then the PCR products were purified and sequenced. For 16S rRNA gene fragments, 566bp nucleotide sequences were obtained, and the A, T, G and C contents in this fragment were respectively 35.16%, 34.45%, 26.44% and 18.02%.As for the COI gene fragments, the size was 658bp and the A, T, G and C contents were 36.63%, 26.44%, 20.52% and 16.41% respectively. Analysis of 16S rRNA and COI gene fragments indicated that: 1) the AT content was higher than CG content, which was similar to the results of studies on drosophila, shrimp, crab and other invertebrates; 2) three samples from the test had the completely same nucleotide sequence of 16S rRNA gene fragments, but there were two locis of T/C translation about the sequence of COI gene fragments. In addition, it is found that the graphical phylogenetic tree of 16S gene fragment of eight orabs in five genus is consistent with current systematics of these species.
Portunus trituberculatus
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Abstract DNA sequence data from a variety of mitochondrial and nuclear gene regions are significant components of phylogenetic research in entomology. Polymerase chain reaction (PCR) amplification primers for many gene regions have been developed that are specific to a range of dipteran groups. Here, we review the existing Diptera-specific PCR amplification primers that have been published for 11 mitochondrial and nuclear gene regions: 12S small ribosomal subunit, cytochrome b, cytochrome oxidase c subunit I, 28S ribosomal RNA, alanyl-tRNA synthetase, the carbamoyl phosphate synthase region of CAD, elongation factor-1α, 6-phosphogluconate dehydrogenase, triose phosphate isomerase, white, and wingless. We also have designed in total 94 new PCR amplification primers for use in these same gene regions. Our new primers have been developed and tested using our DNA sequence database of > 1,600 specimens representing 40 families of Diptera. All of the past and newly developed primer sequences are presented in tables, and their locations are shown on gene maps. This combined data will facilitate future molecular phylogenetic research within Diptera.
Primer (cosmetics)
Triosephosphate isomerase
Nuclear gene
MT-RNR1
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Citations (70)
Objective
To determine the optimal primers of cytochrome C oxidase subunit I (CO I) gene for 8 kinds of rodents in natural epidemic focus of plague in Inner Mongolia.
Methods
In Xilingol League and Wulanchabu City of Inner Mongolia, eight kinds of rodents were collected and DNA was extracted; six pairs of targeted primers (F1/R1, F2/R2, F3/R3, F4/R4, VF/VR, F6/R6) were used to amplify the CO I gene of the 8 species by PCR; PCR products were send to biotechnology company for sequencing, and bioinformatics analysis of the sequencing results was conducted.
Results
F3/R3 was the optimal CO I gene primer for Spermophilus dauricus; cocktail primer (VF/VR) was the optimal CO I gene primer for Ochotona daurica and Lagurus przewalskii; F6/R6 was the optimal CO I gene primer for Rhombomys opimus, Phodopus campbelli and Microtus brandti; F2/R2 was the optimal CO I gene primer for Allactaga sibirica, and F1/R1 was the optimal CO I gene primer for Meriones meridianus. CO I gene sequences of the 8 kinds of rodents were compared; DNA barcoding was unique in each rodent and every rodent had differential point.
Conclusions
Different rodent needs its own species-specific primers for CO I gene amplification. Upon amplification of different rodent, cocktail or F6/R6 primers are the first choices, and choose the species-specific primers for particular species to amplify.
Key words:
Plague; Gene amplification; DNA primer
Primer (cosmetics)
plague
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OBJECTIVE To establish a fluorescent multiple amplification system of 16S rRNA and Cytb genes located in mitochondrial DNA for species identification. METHODS A pair of primers of 16S rRNA gene and Cytb gene of the mitochondrial DNA was designed with the software Primer 5.0 to construct a multiple amplification system. The amplified products from human and five species of animals, including cattle, pig, dog, chicken and grass carp were analyzed by 310 Genetic Analyzer. RESULTS The amplified products of these samples showed two peaks. The common one was 358bp and the specific one different in unique species was between 231bp and 256bp. CONCLUSION The multiplex amplification system can exactly distinguish the species of human from five common animals.
Primer (cosmetics)
Multiplex
Identification
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A PCR-RFLP-based method of species identification was considered for 5 Japanese species of Orius Wolff flower bugs, Orius strigicollis (Poppius), O. minutus (Linnaeus), O. sauteri (Poppius), O. nagaii Yasunaga, and O. tantillus (Motschulsky). Nucleotide sequences of the internal transcribed spacer 1 of the nuclear ribosomal gene and a portion of mitochondrial cytochrome oxidase subunit I gene were compared among species and recognition sites of diagnostically useful restriction enzymes were examined. The PCR-RFLP analysis using 108 individuals of 26 laboratory strains confirmed that the 5 species could be correctly identified by banding patterns generated using these DNA regions. Because our PCR primers can amplify DNA fragments from DNA template extracted from both freshly killed insects and dried specimens stored at room conditions, the PCR-RFLP-based method was considered useful for analyses of field populations in which researchers store and accumulate field-collected samples before they perform laboratory examinations.
Anthocoridae
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