Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content
Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content
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DOI：
10.1016/j.ab.2017.05.011
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发表时间：
2017-08-15
影响因子：
2.9
通讯作者：
Ochiai, Hiroshi
中科院分区：
文献类型：
--
作者：
Ishihara, Satoru;Kotomura, Naoe;Ochiai, Hiroshi
连接介导的聚合酶链反应（LM-PCR）是用于扩增DNA片段池的常用技术。在此，设计了由背靠背方向的两个引物序列组成的双链寡核苷酸作为LM-PCR的衔接子。当DNA片段与该衔接子连接时，片段以随机取向夹在两个衔接子之间。在随后的PCR中，与在每个末端连接到相同引物序列的产物相比，在每个末端连接到衔接子的相对侧（即连接到不同引物序列）的连接产物被优先扩增。与传统的LM-PCR衔接子相比，该衔接子在LM-PCR中的使用减少了高GC含量的底物DNA对PCR的损害。这一结果表明，我们的方法有可能有助于减少由底物DNA中序列背景的固有性质引起的扩增偏倚。用改良的LM-PCR成功地扩增了从使用特定形式的组蛋白H3的下拉的染色质免疫沉淀测定获得的DNA制备物，并且扩增产物可用作荧光原位杂交分析中的探针。(C)2017爱思唯尔公司All rights reserved.
Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. (C) 2017 Elsevier Inc. All rights reserved.