Sinnett D, Flint A, Lalande M
Genetics Division, Children's Hospital, Boston, MA 02115.
Nucleic Acids Res. 1993 Jul 11;21(14):3227-32. doi: 10.1093/nar/21.14.3227.
Studies on the temporal order of DNA replication are difficult due to the lack of sensitivity of methods available for replication kinetic analysis. To overcome problems associated with the current techniques, we propose a PCR-based assay to determine the replication time of any single-copy DNA sequence in complex genomes. Human cells labeled with 5-bromodeoxyuridine (BrdU) were flow sorted, according to their DNA content, at different times after synchronous release from the G1/S phase boundary. The selective removal of newly-replicated BrdU-substituted DNA was achieved by UV light irradiation followed by S1 nuclease treatment. The timing of replication of selected DNA sequences (housekeeping, tissue-specific, and non-coding loci) was determined by polymerase chain reaction (PCR) amplification using appropriate primers. DNA sequences localized in inactive replication units allowed amplification whereas those that have replicated will not be amplified by PCR. Using this sensitive and quantitative assay the replication kinetic analysis of a number of different DNA sequences can be performed from a single sorting experiment.
由于用于复制动力学分析的现有方法缺乏灵敏度，对DNA复制时间顺序的研究存在困难。为了克服与当前技术相关的问题，我们提出了一种基于PCR的检测方法，以确定复杂基因组中任何单拷贝DNA序列的复制时间。用5-溴脱氧尿苷（BrdU）标记的人类细胞在从G1/S期边界同步释放后的不同时间，根据其DNA含量进行流式分选。通过紫外线照射，然后进行S1核酸酶处理，实现对新复制的BrdU取代DNA的选择性去除。使用合适的引物，通过聚合酶链反应（PCR）扩增来确定所选DNA序列（管家基因、组织特异性基因和非编码基因座）的复制时间。位于非活性复制单元中的DNA序列允许扩增，而那些已经复制的序列则不会被PCR扩增。使用这种灵敏且定量的检测方法，可以从单个分选实验中对许多不同的DNA序列进行复制动力学分析。
