PMID: 9186942May 1, 1997Paper

Amplification and cloning of complete enterovirus genomes by long distance PCR

Journal of Virological Methods
A M LindbergS Johansson

Abstract

A method for amplification and cloning of complete enterovirus cDNA genomes is described. Viral RNA was reverse transcribed using an optimized protocol and a reverse transcriptase with reduced RNase H activity. Amplicons corresponding to complete genomes of 14 prototype strains of group B coxsackieviruses and echoviruses were amplified using oligonucleotide primers derived from the Coxsackievirus B3 genomic sequence of the 5' and 3' ends and a mixture of thermostable DNA polymerases. Coxsackievirus B2 amplicon was then cloned and the terminal sequences of the insert were determined. Lipofection of individual clones resulted in productive. Coxsackievirus B2 infection. The method described makes it possible to obtain large amounts of complete enterovirus cDNAs and simplifies the construction of infectious full-length cDNA clones. Successful amplification of all enterovirus prototype strains tested emphasizes the general use of the method described, which provides a rapid and efficient alternative to traditional cloning strategies.

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Citations

Apr 28, 2000·Journal of Virological Methods·A DekkerR J Moormann
Sep 29, 1999·Veterinary Microbiology·F M McCarthyJ S Mattick
Jul 16, 2010·Journal of Virology·Maria GullbergA Michael Lindberg
Jan 15, 2004·Journal of Virology·Nicole G NewcombeDarren R Shafren
Jun 8, 2010·Virology Journal·Jian-Feng HanCheng-Feng Qin
Jun 21, 2005·Virus Research·Charlotta PolacekA Michael Lindberg
May 23, 2001·The Journal of General Virology·C Polacek, A M Lindberg
May 26, 2015·Virus Research·Wangheng HouNingshao Xia
Mar 24, 2016·PloS One·Dhanasekaran GovindarajanAndrew J Bett
Feb 25, 2006·Journal of Virological Methods·Leandro R JonesE Laura Weber
Apr 5, 2007·Nature Protocols·Sébastien F EmonetXavier de Lamballerie
Oct 24, 2003·The Journal of General Virology·Nicole G NewcombeDarren R Shafren

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