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 591 19 |
| Ultima descărcare din IBN: 2024-02-18 02:46 |
| Căutarea după subiecte similare conform CZU |
| 577.2.08 (9) |
| Bazele materiale ale vieții. Biochimie. Biologie moleculară. Biofizică (664) |
 SM ISO690:2012MITIN, Valentin, MITINA, Irina. Some aspects of primer design for real time pcr with sybr green as a dye. In: Journal of Engineering Sciences, 2020, vol. 27, nr. 4, pp. 191-196. ISSN 2587-3474. DOI: https://doi.org/10.5281/zenodo.4296193 |
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| Journal of Engineering Sciences | ||||||
| Volumul 27, Numărul 4 / 2020 / ISSN 2587-3474 /ISSNe 2587-3482 | ||||||
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| DOI:https://doi.org/10.5281/zenodo.4296193 | ||||||
| CZU: 577.2.08 | ||||||
| Pag. 191-196 | ||||||
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This work considers the sequence of steps for designing primers for real time qPCR analysis with SYBR® Green as fluorescent dye. For primer design, we used Primer-Blast software. As an example, we design primers to fum6 gene, a member of fumonisin biosynthetic gene cluster. The importance of a balance between specificity and efficiency of primers obtained with the help of Primer-Blast software is discussed. Challenges of obtaining specific primers are considered, possible solutions are suggested. BLAST analysis of the primers is discussed, potential approaches to choosing an optimal primer pair are considered. The testing results of the designed primers to Fusarium verticillioides fum6 gene in real-time PCR reaction are shown. We used the designed primer pair in a qPCR reaction and analyzed the amplification curve and melting curve of the amplicon. The shape of the amplification curve reflects the kinetics of PCR reaction and gives some approximation about primer efficiency. Melt curve analysis gives the information on primer specificity, with a single melt peak corresponding to a single PCR amplicon. |
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| Cuvinte-cheie Primer-BLAST, Primer3, qPCR, BLAST, Fusarium, Primer-BLAST, Primer3, qPCR, BLAST, Fusarium |
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For primer design, we used Primer-Blast software. As an example, we design primers to fum6 gene, a member of fumonisin biosynthetic gene cluster. The importance of a balance between specificity and efficiency of primers obtained with the help of Primer-Blast software is discussed. Challenges of obtaining specific primers are considered, possible solutions are suggested. BLAST analysis of the primers is discussed, potential approaches to choosing an optimal primer pair are considered. The testing results of the designed primers to Fusarium verticillioides fum6 gene in real-time PCR reaction are shown. We used the designed primer pair in a qPCR reaction and analyzed the amplification curve and melting curve of the amplicon. The shape of the amplification curve reflects the kinetics of PCR reaction and gives some approximation about primer efficiency. Melt curve analysis gives the information on primer specificity, with a single melt peak corresponding to a single PCR amplicon.</p></cfAbstr> <cfAbstr cfLangCode='RO' cfTrans='o'><p>În această lucrare se analizează etapele proiectării primerilor pentru reacția de polimerizare în lanț în timp real cu utilizarea colorantului fluoriscent SYBR® Green. Pentru proiectarea primerilor s-a folosit programul Primer-Blast. În calitate de exemplu, se examinează proiectarea primerilor pentru gena Fusarium verticillioides fum6 în reacția de polimerizare în lanț în timp real. Se pun în discuție aspecte ale analizei BLAST, în scopul optimizării proiectării primerilor. S-a folosit o pereche de primeri într-o reacție qPCR și s-a analizat curba de amplificare și curba de topire a ampliconului. Forma curbei de amplificare reflectă cinetica reacției PCR și ne oferă informații despre eficiența aproximativă a primerilor. 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