tetano
Editor, Senior Moderator
Sci Rep. 2019 Jun 5;9(1):8318. doi: 10.1038/s41598-019-44813-z.
[h=1]Development of a rapid, simple and efficient one-pot cloning method for a reverse genetics system of broad subtypes of influenza A virus.[/h] Choi WS[SUP]1[/SUP], Jeong JH[SUP]1[/SUP], Lloren KKS[SUP]1[/SUP], Ahn SJ[SUP]1[/SUP], Antigua KJC[SUP]1[/SUP], Kim YI[SUP]1[/SUP], Si YJ[SUP]1[/SUP], Baek YH[SUP]1[/SUP], Choi YK[SUP]2[/SUP], Song MS[SUP]3[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The reverse genetics (RG) system of influenza A viruses is well established. However, the conventional sequence-dependent method for cloning influenza genome segments is time-consuming and requires multiple processes (eg. enzyme digestion and ligation) and exhibits low cloning efficiency compared to the sequence-independent cloning method. In this study, we improved influenza genome cloning into the pHW2000 vector for an RG system by incorporating a sequence-independent circular polymerase extension cloning (CPEC) approach which requires only 2 steps (reverse transcription and one-pot CPEC-PCR) and takes about 4 hours before the transformation. The specifically designed viral gene and vector primers used for CPEC-PCR have improved cloning efficiency ranging from 63.6 to 100% based on the results of gene-specific colony PCR which was additionally confirmed by enzyme digestion. We successfully cloned all genes from broad subtypes of influenza A viruses (H1-H12, N1-N9) and rescued by the RG system. Our results demonstrate that this method-one-Pot cloning for influenza A virus-was efficient in terms of required time and cloning rate. In conclusion, the novel cloning method for influenza A virus will contribute to a significant reduction in the time required for genetic studies of emerging influenza viruses.
PMID: 31165766 DOI: 10.1038/s41598-019-44813-z
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[h=1]Development of a rapid, simple and efficient one-pot cloning method for a reverse genetics system of broad subtypes of influenza A virus.[/h] Choi WS[SUP]1[/SUP], Jeong JH[SUP]1[/SUP], Lloren KKS[SUP]1[/SUP], Ahn SJ[SUP]1[/SUP], Antigua KJC[SUP]1[/SUP], Kim YI[SUP]1[/SUP], Si YJ[SUP]1[/SUP], Baek YH[SUP]1[/SUP], Choi YK[SUP]2[/SUP], Song MS[SUP]3[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The reverse genetics (RG) system of influenza A viruses is well established. However, the conventional sequence-dependent method for cloning influenza genome segments is time-consuming and requires multiple processes (eg. enzyme digestion and ligation) and exhibits low cloning efficiency compared to the sequence-independent cloning method. In this study, we improved influenza genome cloning into the pHW2000 vector for an RG system by incorporating a sequence-independent circular polymerase extension cloning (CPEC) approach which requires only 2 steps (reverse transcription and one-pot CPEC-PCR) and takes about 4 hours before the transformation. The specifically designed viral gene and vector primers used for CPEC-PCR have improved cloning efficiency ranging from 63.6 to 100% based on the results of gene-specific colony PCR which was additionally confirmed by enzyme digestion. We successfully cloned all genes from broad subtypes of influenza A viruses (H1-H12, N1-N9) and rescued by the RG system. Our results demonstrate that this method-one-Pot cloning for influenza A virus-was efficient in terms of required time and cloning rate. In conclusion, the novel cloning method for influenza A virus will contribute to a significant reduction in the time required for genetic studies of emerging influenza viruses.
PMID: 31165766 DOI: 10.1038/s41598-019-44813-z
Free full text