Author: Lee, Charlie Wah Heng; Koh, Chee Wee; Chan, Yang Sun; Aw, Pauline Poh Kim; Loh, Kuan Hon; Han, Bing Ling; Thien, Pei Ling; Nai, Geraldine Yi Wen; Hibberd, Martin L.; Wong, Christopher W.; Sung, Wing-Kin
Title: Large-scale evolutionary surveillance of the 2009 H1N1 influenza A virus using resequencing arrays Document date: 2010_2_25
ID: 1rhy8td0_28
Snippet: Due to the small amount of virus present in samples relative to human or cell-line total RNA, it was necessary to amplify the viral RNA through PCR. We employed a combination of sequence-specific and random PCR approaches using LOMA-optimized primers as previously described (11) . The addition of random primers ensured complete genome amplification, even if mutations were present at the specific-primer binding sites. PCR conditions were optimized.....
Document: Due to the small amount of virus present in samples relative to human or cell-line total RNA, it was necessary to amplify the viral RNA through PCR. We employed a combination of sequence-specific and random PCR approaches using LOMA-optimized primers as previously described (11) . The addition of random primers ensured complete genome amplification, even if mutations were present at the specific-primer binding sites. PCR conditions were optimized by conducting five duplicate hybridizations of the same virus sample cultured from a patient sample under different PCR conditions. The optimized method was then tested on RNA isolated directly from nasal swabs obtained from the same patient and from virus grown in cell culture. Microarray sequences generated from these replicate experiments were compared with capillary sequencing to estimate sequencing accuracy.
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