Chang K S, Vyas R C, Deaven L L, Trujillo J M, Stass S A, Hittelman W N
Department of Laboratory Medicine, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Genomics. 1992 Feb;12(2):307-12. doi: 10.1016/0888-7543(92)90378-6.
We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.
我们已经建立了一种通过接头/衔接子连接和聚合酶链反应（PCR）来扩增和获取大量染色体特异性DNA的方法。从经流式细胞术分选的17号和21号染色体中分离出的少量DNA用MboI进行消化，与接头/衔接子连接，然后进行35个循环的PCR。使用该程序，可以获得20微克的染色体特异性DNA。使用先前定位到17号和21号染色体的几种DNA探针进行的Southern印迹分析表明，这些基因序列存在于扩增的染色体特异性DNA中。从第一轮PCR扩增获得的少量染色体特异性DNA被用于第二轮、第三轮和第四轮PCR（30个循环）的DNA扩增，并且仍然可以检测到特异性DNA序列。使用这些染色体特异性DNA探针进行的荧光原位杂交清楚地显示了与指定染色体的杂交信号。我们表明，通过荧光原位杂交，PCR扩增的17号染色体特异性DNA可用于检测急性早幼粒细胞白血病中t(15;17)的非随机染色体易位。
