Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL), a new method to detect translocations in leukemia.
Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL), a new method to detect translocations in leukemia.
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DOI：
10.1371/journal.pone.0104419
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Slany RK
中科院分区：
文献类型：
--
作者：
Fueller E;Schaefer D;Fischer U;Krell PF;Stanulla M;Borkhardt A;Slany RK
在这里，我们提出了一种新的方法“基因组反向PCR探索连接断点”（GIPFEL），允许敏感检测复发性染色体易位。该技术利用有限量的DNA作为起始材料，并依赖于对独特DNA序列的基于PCR的定量，所述独特DNA序列通过来自易位携带细胞的限制性基因组DNA的环状连接而产生。因为询问了完整的潜在断裂点区域，所以不需要个体特异性染色体间融合位点的先验知识。我们验证了GIPFEL用于与儿童白血病相关的五种最常见的基因融合（MLL-AF 4、MLL-AF 9、MLL-ENL、ETV 6-RUNX 1和TCF 3-PBX 1）。开发了限制性消化、纯化、连接、去除线性片段和沉淀富集环状DNA的工作流程。GIPFEL允许检测低至10 - 4稀释度的易位特异性特征序列，这接近理论极限。在一项利用来自细胞系和144名B前体ALL相关易位儿童的DNA的盲法原理验证研究中，该方法具有100%特异性，无假阳性结果。t（4;11）、t（9;11）和t（11;19）的敏感性分别为83%、65%和24%。易位t（12;21）在64%和t（1;19）在39%的情况下被正确检测。与其他方法相比，GIPFEL的特点使其对前瞻性研究特别有吸引力。
Here we present a novel method “Genomic inverse PCR for exploration of ligated breakpoints” (GIPFEL) that allows the sensitive detection of recurrent chromosomal translocations. This technique utilizes limited amounts of DNA as starting material and relies on PCR based quantification of unique DNA sequences that are created by circular ligation of restricted genomic DNA from translocation bearing cells. Because the complete potential breakpoint region is interrogated, a prior knowledge of the individual, specific interchromosomal fusion site is not required. We validated GIPFEL for the five most common gene fusions associated with childhood leukemia (MLL-AF4, MLL-AF9, MLL-ENL, ETV6-RUNX1, and TCF3-PBX1). A workflow of restriction digest, purification, ligation, removal of linear fragments and precipitation enriching for circular DNA was developed. GIPFEL allowed detection of translocation specific signature sequences down to a 10−4 dilution which is close to the theoretical limit. In a blinded proof-of-principle study utilizing DNA from cell lines and 144 children with B-precursor-ALL associated translocations this method was 100% specific with no false positive results. Sensitivity was 83%, 65%, and 24% for t(4;11), t(9;11) and t(11;19) respectively. Translocation t(12;21) was correctly detected in 64% and t(1;19) in 39% of the cases. In contrast to other methods, the characteristics of GIPFEL make it particularly attractive for prospective studies.
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影响因子：
20.3
作者：
Felix, CA;Kim, CS;Rappaport, EF
通讯作者：
Rappaport, EF
影响因子：
20.3
作者：
Schrappe, Martin;Valsecchi, Maria Grazia;Conter, Valentino
通讯作者：
Conter, Valentino
DOI：
10.1073/pnas.94.21.11583
发表时间：
1997-10-14
影响因子：
11.1
作者：
Megonigal, MD;Rappaport, EF;Felix, CA
通讯作者：
Felix, CA
影响因子：
20.3
作者：
Lausten-Thomsen, Ulrik;Madsen, Hans Ole;Schmiegelow, Kjeld
通讯作者：
Schmiegelow, Kjeld
影响因子：
20.3
作者：
Conter, Valentino;Bartram, Claus R.;Schrappe, Martin
通讯作者：
Schrappe, Martin