Troutt A B, McHeyzer-Williams M G, Pulendran B, Nossal G J
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9823-5. doi: 10.1073/pnas.89.20.9823.
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.
已开发出一种简单、高效且灵敏的技术，用于扩增编码5'端序列未知分子的cDNA。在这种连接锚定PCR中，T4 RNA连接酶用于将“锚定”寡核苷酸共价连接到第一链cDNA上。然后使用一种对锚定序列特异的PCR引物和另一种对感兴趣分子内的序列特异的引物，对这些锚定的cDNA进行扩增。锚定寡核苷酸经过专门设计，以利于对所得PCR产物进行后续分析和克隆。这个三阶段过程不需要在步骤之间纯化产物，并且避免了许多与已建立的锚定PCR方案相关的技术难题。通过扩增特定的IgG1 cDNA证明了连接锚定PCR的有效性；相当于仅100个细胞的总RNA产生了预期的PCR产物。
