THE STUDY OF PROKARYOTIC GENE EXPRESSION
One of the methods to evaluate the level of gene expression is a real-time quantitative polymerase chain reaction (qPCR). Interest in the study of molecular mechanisms of gene expression and its evaluation in prokaryotes is due to the lack of research on this issue and a number of methodological pro...
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| Format: | Article |
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The V.M. Gorbatov All-Russian Meat Research Institute
2018-07-01
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| Series: | Теория и практика переработки мяса |
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| Online Access: | https://www.meatjournal.ru/jour/article/view/91 |
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| author | Mihail Yu. Minaev Anzhelika A. Makhova |
| author_facet | Mihail Yu. Minaev Anzhelika A. Makhova |
| author_sort | Mihail Yu. Minaev |
| collection | DOAJ |
| description | One of the methods to evaluate the level of gene expression is a real-time quantitative polymerase chain reaction (qPCR). Interest in the study of molecular mechanisms of gene expression and its evaluation in prokaryotes is due to the lack of research on this issue and a number of methodological problems. The paper presents a study of gene expression mechanism in prokaryotes evidence from Aeromonas salmonicida AS1 gyrase B and collagenase genes. As a result of the research, Random primer and oligo (dT) primer (two 3’-terminal nucleotides of the primer complementary to stop codon nucleotides of the transcribed DNA sequence) with anchor and adapter of our own design were tested, which are used in the reaction of reverse transcription. The use of oligo (dT) primer became possible only after polyadenylation of extracted RNA using special poly-A polymerase kit. It is determined that the developed protocol of reverse transcription (RT) using oligo (dT) primer and adapter with certain sequence on its 5’-terminus designed for further annealing of the reverse primer during real-time PCR along with preliminary polyadenylation of RNA excludes specific amplification of the background genomic DNA. This technique may be applied in evaluating the expression level of low-expression genes when high background genomic DNA content is found in the RNA sample, e.g. at the end of logarithmic growth of prokaryotic cells.ContributionAll authors bear responsibility for the work and presented data. All authors made an equal contribution to the work. Minaev M. Yu. developed scientific and methodological approaches to work, determined the scope of research, analyzed the data obtained, performed the narrative and corrected it in final. Makhova A.A. selected research objects, carried out RNA extraction, reverse transcription and PCR analysis, performed the narrative part. The authors were equally involved in writing the manuscript and bear the equal responsibility for plagiarism.Conflict of interestThe authors declare no conflict of interest. |
| format | Article |
| id | doaj-art-fc7cd08c36f84d35878e6f79802f1821 |
| institution | Kabale University |
| issn | 2414-438X 2414-441X |
| language | English |
| publishDate | 2018-07-01 |
| publisher | The V.M. Gorbatov All-Russian Meat Research Institute |
| record_format | Article |
| series | Теория и практика переработки мяса |
| spelling | doaj-art-fc7cd08c36f84d35878e6f79802f18212025-08-20T03:59:57ZengThe V.M. Gorbatov All-Russian Meat Research InstituteТеория и практика переработки мяса2414-438X2414-441X2018-07-0132405210.21323/2414-438X-2018-3-2-40-5271THE STUDY OF PROKARYOTIC GENE EXPRESSIONMihail Yu. Minaev0Anzhelika A. Makhova1V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of SciencesV.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of SciencesOne of the methods to evaluate the level of gene expression is a real-time quantitative polymerase chain reaction (qPCR). Interest in the study of molecular mechanisms of gene expression and its evaluation in prokaryotes is due to the lack of research on this issue and a number of methodological problems. The paper presents a study of gene expression mechanism in prokaryotes evidence from Aeromonas salmonicida AS1 gyrase B and collagenase genes. As a result of the research, Random primer and oligo (dT) primer (two 3’-terminal nucleotides of the primer complementary to stop codon nucleotides of the transcribed DNA sequence) with anchor and adapter of our own design were tested, which are used in the reaction of reverse transcription. The use of oligo (dT) primer became possible only after polyadenylation of extracted RNA using special poly-A polymerase kit. It is determined that the developed protocol of reverse transcription (RT) using oligo (dT) primer and adapter with certain sequence on its 5’-terminus designed for further annealing of the reverse primer during real-time PCR along with preliminary polyadenylation of RNA excludes specific amplification of the background genomic DNA. This technique may be applied in evaluating the expression level of low-expression genes when high background genomic DNA content is found in the RNA sample, e.g. at the end of logarithmic growth of prokaryotic cells.ContributionAll authors bear responsibility for the work and presented data. All authors made an equal contribution to the work. Minaev M. Yu. developed scientific and methodological approaches to work, determined the scope of research, analyzed the data obtained, performed the narrative and corrected it in final. Makhova A.A. selected research objects, carried out RNA extraction, reverse transcription and PCR analysis, performed the narrative part. The authors were equally involved in writing the manuscript and bear the equal responsibility for plagiarism.Conflict of interestThe authors declare no conflict of interest.https://www.meatjournal.ru/jour/article/view/91gene expressionprokaryotespcrassay |
| spellingShingle | Mihail Yu. Minaev Anzhelika A. Makhova THE STUDY OF PROKARYOTIC GENE EXPRESSION Теория и практика переработки мяса gene expression prokaryotes pcr assay |
| title | THE STUDY OF PROKARYOTIC GENE EXPRESSION |
| title_full | THE STUDY OF PROKARYOTIC GENE EXPRESSION |
| title_fullStr | THE STUDY OF PROKARYOTIC GENE EXPRESSION |
| title_full_unstemmed | THE STUDY OF PROKARYOTIC GENE EXPRESSION |
| title_short | THE STUDY OF PROKARYOTIC GENE EXPRESSION |
| title_sort | study of prokaryotic gene expression |
| topic | gene expression prokaryotes pcr assay |
| url | https://www.meatjournal.ru/jour/article/view/91 |
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