LIGATION-ANCHORED PCR - A SIMPLE AMPLIFICATION TECHNIQUE WITH SINGLE-SIDED SPECIFICITY
LIGATION-ANCHORED PCR - A SIMPLE AMPLIFICATION TECHNIQUE WITH SINGLE-SIDED SPECIFICITY
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DOI：
10.1073/pnas.89.20.9823
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发表时间：
1992-10-15
影响因子：
11.1
通讯作者：
NOSSAL, GJV
中科院分区：
文献类型：
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作者：
TROUTT, AB;MCHEYZERWILLIAMS, MG;NOSSAL, GJV
一种简单、有效和灵敏的技术已经被开发用于扩增编码具有未知序列的5'区的分子的cDNA。在该连接锚定PCR中，使用T4 RNA连接酶将“锚”寡核苷酸共价连接至第一链cDNA。然后通过使用一种对锚特异的PCR引物和另一种对感兴趣的分子内的序列特异的PCR引物来扩增这些锚定的cDNA。特别设计了锚寡核苷酸以便于随后分析和克隆所得PCR产物。这种三阶段程序不需要在步骤之间纯化产物，并且避免了与已建立的锚定PCR方案相关的许多技术困难。连接锚定PCR的有效性证明了一个特定的IgG 1 cDNA的扩增;总RNA相当于少至100个细胞产生预期的PCR产物。
A simple, efficient, and sensitive technique has been developed for amplification of cDNAs encoding molecules with 5' regions of unknown sequence. In this ligation-anchored PCR, T4 RNA ligase is used to covalently link an "anchor" oligonucleotide to first-strand cDNAs. These anchored cDNAs are then amplified by using one PCR primer specific for the anchor and another specific for a sequence within the molecule of interest. The anchor oligonucleotide has been especially designed to facilitate subsequent analysis and cloning of the resultant PCR products. This three-stage procedure does not require purification of product between steps and avoids many of the technical difficulties associated with established anchored PCR protocols. The efficacy of ligation-anchored PCR was demonstrated by amplification of a specific IgG1 cDNA; total RNA equivalent to as few as 100 cells yielded the expected PCR product.