Horsthemke B, Claussen U, Hesse S, Lüdecke H J
Institut für Humangenetik, Universitätsklinikum Essen, Germany.
PCR Methods Appl. 1992 May;1(4):229-33. doi: 10.1101/gr.1.4.229.
Vertebrate DNA contains a small fraction of unmethylated CpG-rich DNA sequences, many of which include the 5' end of a gene. This fraction can be detected by its cleavage to tiny fragments with the methylation-sensitive restriction enzyme HpaII. Thus, the isolation of HpaII tiny fragments (HTFs) from a specific chromosome region may be a useful approach for making an inventory of the genes contained in it. Using microdissection, we have isolated DNA from human chromosome band 8q24.1. The DNA was digested with HpaII, ligated to a ClaI-cut pUC plasmid, and amplified with Taq DNA polymerase and the standard M13/pUC forward and reverse sequencing primers. The amplification products were used to construct an HTF library, which is enriched for CpG-rich single-copy clones.
脊椎动物的DNA含有一小部分未甲基化的富含CpG的DNA序列，其中许多序列包含基因的5'端。这一部分可以通过用甲基化敏感的限制性内切酶HpaII切割成小片段来检测。因此，从特定染色体区域分离HpaII小片段（HTF）可能是一种有用的方法，用于对其中所含基因进行编目。通过显微切割，我们从人类染色体带8q24.1中分离出了DNA。该DNA用HpaII消化，连接到经ClaI切割的pUC质粒上，并用Taq DNA聚合酶以及标准的M13/pUC正向和反向测序引物进行扩增。扩增产物用于构建一个HTF文库，该文库富含富含CpG的单拷贝克隆。
