Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.
Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.
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Panhandle PCR for cDNA：一种快速分离涉及未知伴侣基因的 MLL 融合转录本的方法。
DOI：
10.1073/pnas.150241797
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发表时间：
2000
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;Wilson,RB;Jones,DH;Whitlock,JA;Ortega,JA;Slater,DJ;Nowell,PC;Felix,CA
确定MLL基因在染色体11q23的易位对于白血病的特征和治疗具有重要意义。然而，细胞遗传学分析并不总是发现易位，而且MLL的许多配对基因使分子检测变得困难。我们开发了cDNAPanHandle聚合酶链式反应来鉴定Der(11)转录本，而不考虑伴侣基因。通过用5‘端含有5’MLL断点簇区编码序列和3‘端随机六聚体的寡核苷酸逆转录第一链cDNA，已知的MLL序列被连接到未知的配对序列上。这使得茎环模板的形成具有嵌合转录本在环中的融合点，并在双边PCR中使用MLL引物。通过检测MV4-11细胞系中已知的融合转录本和正常MLL等位基因的转录本进行了验证。然后用cDNAPanHandle聚合酶链式反应对两例核型正常、配对基因未知的治疗相关性急性髓系白血病患者的融合转录本进行了鉴定。1例MLL与AF-10融合，1例MLL与ELLIN融合。另外，该方法还可以检测到剪接转录本和外显子扰乱。核型正常的白血病可能含有带有多种配对基因的隐匿性MLL易位。在融合转录本中检测MLL易位和确定未知的合作伙伴序列是有用的。
Identifying translocations of theMLLgene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes ofMLLmake molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5′MLLbreakpoint cluster region at the 5′ ends and random hexamers at the 3′ ends, knownMLLsequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use ofMLLprimers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normalMLLallele in the cell line MV4–11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion ofMLLwithAF-10in one case and a fusion ofMLLwithELLin the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations ofMLLwith a variety of partner genes. cDNA panhandle PCR is useful for identifyingMLLtranslocations and determining unknown partner sequences in the fusion transcripts.