Nucleic acid spot hybridization: rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA.
Nucleic acid spot hybridization: rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA.
复制标题
核酸点杂交：快速定量筛选淋巴细胞系中的 Epstein-Barr 病毒 DNA。
DOI：
10.1073/pnas.77.11.6851
复制
发表时间：
1980
影响因子：
11.1
通讯作者：
G. Miller
中科院分区：
文献类型：
--
作者：
J. Brandsma;G. Miller
描述了一种简单的核酸杂交方法，用于快速筛选大量真核细胞样品的EB病毒（EBV）DNA含量。将整个细胞点在硝酸纤维素滤膜上，使其DNA变性并固定在滤膜上。将所得DNA斑点与切口翻译的EBV DNA杂交，并通过放射自显影和闪烁计数监测杂交程度。细胞连续稀释液的统计分析允许通过参考已知标准品（如Raji细胞或纯病毒DNA和未感染淋巴细胞的人工混合物）定量估计其病毒基因组含量。该方法的灵敏度为5至50 pg的病毒DNA。利用这种方法，我们能够选择高产量的EBV DNA的亚克隆，并确定从培养的细胞中收获EBV DNA的最佳时间。斑点杂交应允许筛选任何细胞群体或流体中存在的DNA序列，放射性同位素标记的探针是可用的。
A simple nucleic acid hybridization method to screen numerous samples of eukaryotic cells rapidly for their Epstein-Barr virus (EBV) DNA content is described. Whole cells are spotted on nitrocellulose filters and their DNA is denatured and fixed to the filter. The resultant DNA spots are hybridized to nick-translated EBV DNA and the extent of hybridization is monitored by autoradiography and scintillation counting. Statistical analysis of serial dilutions of cells permits their viral genome content to be estimated quantitatively by reference to a known standard, such as Raji cells or an artificial mixture of pure viral DNA and uninfected lymphocytes. The sensitivity of the method is between 5 and 50 pg of viral DNA. With this method we are able to select subclones that are high produces of EBV DNA and to identify the optimal time for harvest of EBV DNA from cultured cells. Spot hybridization should permit any cell population or fluid to be screened for the presence of a DNA sequence for which a radioisotopically labeled probe is available.