Wooddell C I, Burgess R R
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706, USA.
Genome Res. 1996 Sep;6(9):886-92. doi: 10.1101/gr.6.9.886.
Photoactivatable DNA analogs have been incorporated enzymatically into DNA and used to map the locations of polypeptides in protein complexes bound to DNA. We have developed a procedure for generating long primers from short oligodeoxyribonucleotides (oligos) to incorporate DNA cross-linkers at specific sites within either strand of DNA probes of < or = 206 bp. Single-stranded DNA molecules of 52-206 nucleotides in length were generated by asymmetric polymerase chain reactions (aPCR), using an excess of one short sense-strand primer to be extended and a limiting amount of each short antisense primer that is complementary to and defines the 3' end of the long primer to be generated. The noncross-linking strand of the DNA probe was also generated by aPCR from the DNA sequence of interest. The long primers were annealed to the full-length noncross-linking DNA strand to form a partially double-stranded DNA. Cross-linking analogs and radioactive deoxyribonucleotides (dNTPs), followed by normal dNTPs, were enzymatically incorporated onto the long primers to form the double-stranded DNA cross-linking probes. This method is reproducible and avoids many of the difficulties encountered by other published methods.
可光激活的DNA类似物已通过酶促方式掺入DNA中，并用于绘制与DNA结合的蛋白质复合物中多肽的位置。我们已经开发出一种从短寡脱氧核糖核苷酸（寡核苷酸）生成长引物的方法，以便在长度小于或等于206bp的DNA探针的任一条链内的特定位点掺入DNA交联剂。通过不对称聚合酶链反应（aPCR）生成长度为52 - 206个核苷酸的单链DNA分子，使用过量的一种待延伸的短正义链引物和有限量的每种与待生成的长引物的3'端互补并定义其的短反义引物。DNA探针的非交联链也通过aPCR从感兴趣的DNA序列生成。将长引物与全长非交联DNA链退火以形成部分双链DNA。然后将交联类似物和放射性脱氧核糖核苷酸（dNTP），接着是正常的dNTP，酶促掺入到长引物上以形成双链DNA交联探针。该方法具有可重复性，并且避免了其他已发表方法所遇到的许多困难。
