Bohlander S K, Espinosa R, Le Beau M M, Rowley J D, Díaz M O
Section of Hematology/Oncology, University of Chicago, Illinois 60637.
Genomics. 1992 Aug;13(4):1322-4. doi: 10.1016/0888-7543(92)90057-y.
We have developed a simple, efficient method by which microdissected material can be amplified directly in the collection container in a few hours. The procedure involves two initial rounds of DNA synthesis with T7 DNA polymerase, using a primer that contains a random pentanucleotide sequence at its 3' end and a defined sequence at its 5' end, followed by PCR amplification with the defined sequence as the primer. The resulting products can be biotinylated and used for fluorescence in situ hybridization (FISH) to confirm their chromosomal location. As few as 17 dissected chromosomal regions provide sufficient material for a specific FISH signal on the appropriate band of metaphase chromosomes. We have obtained a chromosome 6q25-qter-specific painting probe in this way.
我们开发了一种简单、高效的方法，通过该方法可以在几小时内直接在收集容器中对显微切割的材料进行扩增。该程序包括两轮使用T7 DNA聚合酶的初始DNA合成，使用的引物在其3'端含有一个随机的五核苷酸序列，在其5'端含有一个确定的序列，随后以该确定的序列作为引物进行PCR扩增。所得产物可以进行生物素化，并用于荧光原位杂交（FISH）以确认其染色体定位。少至17个显微切割的染色体区域就能为中期染色体的适当条带提供足够的材料以产生特异性FISH信号。我们通过这种方式获得了一个6q25-qter特异性的染色体涂染探针。
