LoPPS: a long PCR product sequencing method for rapid characterisation of long amplicons |
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Authors: | Emonet Sébastien Grard Gilda Brisbarre Nadège Moureau Gregory Temmam Sarah Charrel Rémi de Lamballerie Xavier |
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Affiliation: | Unité des Virus Emergents (EA3292, IFR48, IRD UR034), Faculté de Médecine, 27 boulevard Jean Moulin, 13005 Marseille, France. |
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Abstract: | Here, we propose an optimised protocol (LoPPS, long PCR product sequencing) which allows the fast, cost-attractive, and high-throughput sequencing of long PCR products. LoPPS constitutes an alternative to the primer-walking technology which is expensive and time consuming but remains the current standard procedure. It is based on the ultrasonic shearing, polishing, and cloning of PCR or RT-PCR products and is compatible with 96- or 384-well microplate systems in which bacterial growth, preparation of plasmid DNA, and sequencing can be automated. We present results obtained from 24 different RT-PCR products (2.5-4.8 kbp long) obtained from various RNA viruses and fully sequenced using LoPPS. The method proved to be robust and fast. It was successfully used on a low amount of DNA and allowed each target nucleotide position to be controlled twice or more, with a final cost which is one-third of that of primer-walking. |
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Keywords: | Sequencing PCR RNA viruses LoPPS |
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