Bischofberger N, Ng P G, Webb T R, Matteucci M D
Nucleic Acids Res. 1987 Jan 26;15(2):709-16. doi: 10.1093/nar/15.2.709.
The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.
合成了31聚体5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T、互补的33聚体5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT、40聚体5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G以及互补的42聚体5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC，并研究了它们对EcoRI的反应性。发现31聚体和40聚体分别以与31聚体-33聚体杂交体和40聚体-42聚体杂交体相当的速率被切割。33聚体和42聚体的切割速率低一个数量级。为了排除可能的分子间双链体形成，通过连接将33聚体固定在纤维素上，并使用大肠杆菌DNA聚合酶的Klenow片段用α-32P-dCTP进行标记。EcoRI将这种固定化的寡聚物切割成特定的片段。
