Mills K I, Sproul A M, Ogilvie D, Elvin P, Leibowitz D, Burnett A K
Department of Haematology, Glasgow Royal Infirmary, Scotland, UK.
Leukemia. 1992 May;6(5):481-3.
The polymerase chain reaction (PCR) cannot be used to amplify the breakpoint in the chimaeric BCR-ABL gene in CML and acute leukaemias due to the large variation in the sites of breakpoint in the BCR gene (within a 5.8 kb region) and in the ABL gene (within a 150 kb region). The disease state is usually monitored using RNA-PCR to monitor abnormal transcripts. We have used a new modification of the PCR to amplify breakpoints within zone 3 of the M-bcr. A synthetic oligonucleotide linker, the Vectorette, is ligated to restriction digested DNA, and amplification is carried out between primers for a known target sequence and the Vectorette linker. Three Philadelphia chromosome Ph1-positive CML patients with breakpoints within the ALU region of zone 3 have been amplified and the sequence immediately around the breakpoint determined. The breaks occurred within 70 bp and two were only 14 bp apart. The Vectorette-PCR technique has the potential to rapidly identify and sequence breakpoints, and will enable the design of patient-specific primers to monitor disease progression, particularly following bone marrow transplantation.
由于BCR基因（在5.8 kb区域内）和ABL基因（在150 kb区域内）的断点位置存在很大差异，聚合酶链反应（PCR）不能用于扩增慢性粒细胞白血病（CML）和急性白血病中嵌合性BCR-ABL基因的断点。通常使用RNA-PCR监测异常转录本来监测疾病状态。我们采用了一种新的PCR改良方法来扩增M-bcr第3区的断点。将一种合成的寡核苷酸接头（Vectorette）连接到经限制性酶切的DNA上，并在针对已知靶序列的引物和Vectorette接头之间进行扩增。对3例Ph1阳性CML患者（其断点位于第3区的ALU区域内）进行了扩增，并确定了断点周围的序列。断点发生在70 bp范围内，其中两个断点仅相隔14 bp。Vectorette-PCR技术有潜力快速鉴定断点并进行测序，还能设计针对患者的引物来监测疾病进展，尤其是在骨髓移植后。
