BglII-based panhandle and reverse panhandle PCR approaches increase capability for cloning der(II) and der(other) genomic breakpoint junctions of MLL translocations.
BglII-based panhandle and reverse panhandle PCR approaches increase capability for cloning der(II) and der(other) genomic breakpoint junctions of MLL translocations.
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基于 BglII 的 panhandle 和反向 panhandle PCR 方法提高了克隆 MLL 易位的 der(II) 和 der(other) 基因组断点连接的能力。
DOI：
10.1002/gcc.20336
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发表时间：
2006
期刊：
影响因子：
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通讯作者：
Felix,CarolynA
中科院分区：
文献类型：
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作者：
Robinson,BlaineW;Slater,DianaJ;Felix,CarolynA
扩增未知序列侧翼的已知序列的柄状PCR技术对于MLL基因组断点连接和融合转录物是有用的，因为MLL具有大量的伴侣基因。然而，当含有断点连接的限制性片段太大而不能扩增时，基因组柄状PCR方法受到阻碍。我们设计了一种新的MLL基因组断点连接的柄状PCR方法，通过将MLL序列连接到伴侣基因中的BglII位点，从BglII限制性片段中创建模板。这导致ML及其互补序列在柄中退火，并产生含有断点连接序列的链内环，用于用全部来自MLL的引物进行扩增。Bgl Ⅱ pannhandle PCR检测der（11）断裂点连接是通过将含有Bgl Ⅱ突出端和与MLLexon 7互补序列的磷酸化寡核苷酸连接到Bgl Ⅱ消化的DNA的3′端，并从DNA的有义链形成模板来实现的。在Bgl Ⅱ反向柄状PCR中，将一个含有Bgl Ⅱ突出端和MLL 10外显子反义序列互补序列的磷酸化寡核苷酸连接到Bgl Ⅱ酶切DNA的3′端，并由DNA的反义链形成模板。这些入路可扩增5 ′-MLL-MLLT 4 - 3′和5 ′-AFF 1-MLL-3′断裂点连接。前者是重要的，因为很少有t（6;11）基因组断裂点连接已被测序。BglII柄状PCR方法增加了克隆MLL基因组断裂点连接的可能性，其中在伴侣基因和断裂点位置中存在异质性。© 2006 Wiley利斯公司
Panhandle PCR techniques to amplify known sequence flanked by unknown sequence have been useful forMLLgenomic breakpoint junctions and fusion transcripts becauseMLLhas a large number of partner genes. However, genomic panhandle PCR approaches are impeded when the restriction fragment that contains the breakpoint junction is too large to amplify. We devised new panhandle PCR approaches forMLLgenomic breakpoint junctions that create the template fromBglII restriction fragments by attachingMLLsequence to aBglII site in the partner gene. This leads to the annealing ofMLLand its complement in the handle and creates an intrastrand loop containing the breakpoint junction sequence for amplification with primers all fromMLL. BglII panhandle PCR for der(11) breakpoint junctions was accomplished by ligating a phosphorylated oligonucleotide containing aBglII overhang and sequence complementary toMLLexon 7 to the 3′ ends ofBglII digested DNA, and forming the template from the sense strand of DNA. InBglII reverse panhandle PCR for der(other) breakpoint junctions, a phosphorylated oligonucleotide containing aBglII overhang and the complement of antisense sequence inMLLexon 10 was ligated to the 3′ ends ofBglII digested DNA, and the template was formed from the antisense strand of DNA. These approaches amplified5′‐MLL‐MLLT4‐3′ and5′‐AFF1‐MLL‐3′ breakpoint junctions. The former is significant because few t(6;11) genomic breakpoint junctions have been sequenced.BglII panhandle PCR approaches increase the possibilities for cloningMLLgenomic breakpoint junctions where there is heterogeneity in partner genes and breakpoint locations. © 2006 Wiley‐Liss, Inc.