Hadano S, Watanabe M, Yokoi H, Kogi M, Kondo I, Tsuchiya H, Kanazawa I, Wakasa K, Ikeda J E
Ikeda Genosphere Project, ERATO, JRDC, Tokai University School of Medicine, Kanagawa, Japan.
Genomics. 1991 Oct;11(2):364-73. doi: 10.1016/0888-7543(91)90144-4.
We have developed an argon laser chromosome microdissection technique in conjunction with a polymerase chain reaction (PCR) approach to directly amplify microdissected chromosomes. The single 22-mer primer used in PCR, although unique in sequence (5'-TAGATCTGA-TATCTGAATTCCC-3'), randomly primed and amplified any target DNA. These methods were applied to the distal half of the short arm of human chromosome 4 containing the Huntington disease (HD) locus. Forty-four percent of representative clones from this library identify single-copy DNA sequences. This calculation suggests that the resulting chromosome-specific DNA library contains approximately 600 nonoverlapping sequences with an average size 350 bp at an average spacing of 30 kbp along chromosome 4. This microdissection and PCR cloning procedure is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.
我们结合聚合酶链反应（PCR）方法开发了一种氩激光染色体显微切割技术，用于直接扩增显微切割的染色体。PCR中使用的单一22聚体引物，尽管序列独特（5'-TAGATCTGA-TATCTGAATTCCC-3'），但能随机引发并扩增任何目标DNA。这些方法应用于包含亨廷顿病（HD）基因座的人类4号染色体短臂的远端一半。该文库中44%的代表性克隆鉴定出单拷贝DNA序列。这一计算表明，所得的染色体特异性DNA文库包含约600个非重叠序列，平均大小为350 bp，沿4号染色体平均间距为30 kbp。这种显微切割和PCR克隆程序是一种从单个中期铺片中构建染色体区域特异性DNA文库的简单通用方法。
