Panhandle PCR strategy to amplify MLL genomic breakpoints in treatment-related leukemias
Panhandle PCR strategy to amplify MLL genomic breakpoints in treatment-related leukemias
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DOI：
10.1073/pnas.94.21.11583
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发表时间：
1997-10-14
影响因子：
11.1
通讯作者：
Felix, CA
中科院分区：
文献类型：
--
作者：
Megonigal, MD;Rappaport, EF;Felix, CA
我们用PanHandle PCR从模板中扩增出已知5‘和未知3’序列的基因组DNA，其链内环呈带柄的PAN状。我们用PanHandle PCR克隆了两种儿科治疗相关白血病的MLL基因组断裂点。1例治疗相关的急性髓系白血病患者核型正常，Southern杂交分析显示有单个MLL基因重排，PanHandle PCR扩增出MLL内含子6中1493位的断裂点，体细胞杂交种和辐射杂交种DNA的筛选和白血病细胞的逆转录聚合酶链式反应分析表明，PanHandle PCR检测到MLL内含子6与MLL内含子1中一个未知序列的融合，与部分重复序列一致。在两例病例中，MLL内含子6中的断裂点都在Alu重复序列中，在配对DNA断裂点附近有Alu重复，提示Alu序列与这些重排有关。本研究表明，狭长柄聚合酶链式反应是克隆治疗相关白血病MLL基因组断裂点的有效方法。对其他儿科病例的分析将确定断点分布是否偏离已在成人病例中发现的BCR中3‘分布的倾向。
Panhandle PCR amplifies genomic DNA with known 5' and unknown 3' sequences from a template with an intrastrand loop schematically shaped like a pan with a handle, We used panhandle PCR to clone MLL genomic breakpoints in two pediatric treatment-related leukemias, The karyotype in a case of treatment-related acute lympho blastic leukemia showed the t(4;11) (q21;q23), Panhandle PCR amplified the translocation breakpoint at position 2158 in intron 6 in the 5' MLL breakpoint cluster region (bcr), The karyotype in a case of treatment-related acute myeloid leukemia was normal, but Southern blot analysis showed a single MLL gene rearrangement, Panhandle PCR amplified the breakpoint at position 1493 in MLL intron 6, Screening of somatic cell hybrid and radiation hybrid DNAs by PCR and reverse transcriptase-PCR analysis of the leukemic cells indicated that panhandle PCR identified a fusion of MLL intron 6 with a previously uncharacterized sequence in MLL intron 1, consistent with a partial duplication, In both cases, the breakpoints in the MLL bcr were in Alu repeats, and there were Alu repeats in proximity to the breakpoints in the partner DNAs, suggesting that Alu sequences were relevant to these rearrangements, This study shows that panhandle PCR is an effective method for cloning MLL genomic breakpoints in treatment-related leukemias, Analysis of additional pediatric cases will determine whether breakpoint distribution deviates from the predilection for 3' distribution in the bcr that has been found in adult cases.