一种基于快速逆转录聚合酶链反应的方法，用于分离逆转录病毒整合位点侧翼的互补DNA片段。
A rapid RT-PCR based method to isolate complementary DNA fragments flanking retrovirus integration sites.
作者信息
Valk P J, Joosten M, Vankan Y, Löwenberg B, Delwel R
机构信息
Institute of Hematology, Erasmus University Rotterdam, The Netherlands.
出版信息
Nucleic Acids Res. 1997 Nov 1;25(21):4419-21. doi: 10.1093/nar/25.21.4419.
Abstract
Proto-oncogenes in retrovirally induced myeloid mouse leukemias are frequently activated following retroviral insertion. The identification of common virus integration sites (VISs) and isolation of the transforming oncogene is laborious and time consuming. We established a rapid and simple PCR based procedure which facilitates the identification of VISs and novel proto-oncogenes. Complementary DNA fragments adjacent to retrovirus integration sites were selectively isolated by applying a reverse transcriptase (RT) reaction using an oligo(dT)-adaptor primer, followed by PCR using the adaptor sequence and a retrovirus long terminal repeat (LTR) specific primer. Multiple chimeric cDNA fragments suitable for Southern and northern blot analysis were isolated.
摘要
逆转录病毒诱导的小鼠髓系白血病中的原癌基因在逆转录病毒插入后常常被激活。鉴定常见的病毒整合位点（VISs）并分离转化癌基因既费力又耗时。我们建立了一种基于PCR的快速简便方法，有助于鉴定VISs和新型原癌基因。通过使用寡聚（dT）-接头引物进行逆转录酶（RT）反应，选择性地分离与逆转录病毒整合位点相邻的互补DNA片段，随后使用接头序列和逆转录病毒长末端重复序列（LTR）特异性引物进行PCR。分离出了多个适用于Southern和Northern印迹分析的嵌合cDNA片段。
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