Panhandle polymerase chain reaction amplifies MLL genomic translocation breakpoint involving unknown partner gene
Panhandle polymerase chain reaction amplifies MLL genomic translocation breakpoint involving unknown partner gene
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DOI：
10.1182/blood.v90.12.4679.4679_4679_4686
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发表时间：
1997-12-15
期刊：
影响因子：
20.3
通讯作者：
Rappaport, EF
中科院分区：
文献类型：
--
作者：
Felix, CA;Kim, CS;Rappaport, EF
我们使用了一种新的方法，称为锅柄聚合酶链反应（PCR）克隆MLL基因组易位断点的情况下，急性淋巴细胞白血病的婴儿，核型分析是技术上不成功的，并没有显示易位的合作伙伴。Panhandle PCR从具有链内环的DNA模板扩增断裂点5'的已知MLL序列和来自未知配偶体基因的3'序列，所述链内环的形状示意性地像具有柄的盘。7-kb柄状PCR产物在MLL内含子8中含有易位断裂点。伴侣DNA包括独特的非重复序列、Alu和哺乳动物表观LTR-反转录转座子（MaLR）重复序列以及与表达序列标签同源的区域。在白血病相关易位断裂点附近之前尚未发现MaLR序列。非重复序列与已知的MLL伴侣基因不同源。通过PCR和荧光原位杂交技术筛选体细胞杂种和辐射杂种系，将其配偶体DNA定位于染色体4 q21带。逆转录酶-PCR鉴定了MLL-AF-4嵌合mRNA，表明柄状PCR鉴定了MLL与先前未表征的AF-4内含子序列的融合。柄状PCR有助于克隆易位断裂点和鉴定未知的伴侣基因。(C)1997年，美国血液学会。
We used a new approach called panhandle polymerase chain reaction (PCR) to clone an MLL genomic translocation breakpoint in a case of acute lymphoblastic leukemia of infancy in which karyotype analysis was technically unsuccessful and did not show the translocation partner. Panhandle PCR amplified known MLL sequence 5' of the breakpoint and 3' sequence from the unknown partner gene from a DNA template with an intrastrand loop schematically shaped like a pan with a handle. The 7-kb panhandle PCR product contained the translocation breakpoint in MLL intron 8. The partner DNA included unique nonrepetitive sequences, Alu and mammalian apparent LTR-retrotransposon (MaLR) repetitive sequences, and a region of homology to expressed sequence tags. MaLR sequences have not been found before near leukemia-associated translocation breakpoints. The nonrepetitive sequences were not homologous to known partner genes of MLL. Screening of somatic cell hybrid and radiation hybrid lines by PCR and fluorescence in situ hybridization analysis of normal metaphase chromosomes mapped the partner DNA to chromosome band 4q21. Reverse transcriptase-PCR identified an MLL-AF-4 chimeric mRNA, indicating that panhandle PCR identified a fusion of MLL with a previously uncharacterized AF-4 intronic sequence. panhandle PCR facilitates cloning translocation breakpoints and identifying unknown partner genes. (C) 1997 by The American Society of Hematology.