利用纳克量病毒DNA的多重置换扩增对犀牛金龟子浓核病毒的大型双链DNA基因组进行测序。
Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.
作者信息
Wang Yongjie, Kleespies Regina G, Ramle Moslim B, Jehle Johannes A
机构信息
Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate (DLR Rheinpfalz), Breitenweg 71, 67435, Neustadt an der Weinstrasse, Germany.
出版信息
J Virol Methods. 2008 Sep;152(1-2):106-8. doi: 10.1016/j.jviromet.2008.06.003. Epub 2008 Jul 18.
The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.
许多大型双链DNA病毒的基因组序列分析因缺乏足够的样本材料而受到阻碍。在此，我们报告了从仅约10 ng纯化的病毒DNA开始，通过应用基于phi29 DNA聚合酶和抗外切核酸酶的随机六聚体的多重置换扩增（MDA）方法，对独角仙浓核病毒（OrNV）分离株Ma07进行全基因组扩增。扩增得到了约60 μg片段大小达25 kbp的高分子量DNA。使用产物DNA构建了基因组DNA克隆文库。经过8倍测序覆盖后，成功测定了OrNV全基因组的127,615 bp。结果表明，基于MDA的全基因组扩增能够从稀少的病毒样本中快速获取基因组信息。
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