Tamme R, Mills K, Rainbird B, Nornes S, Lardelli M
The University of Adelaide, Australia.
Biotechniques. 2001 Oct;31(4):938-42, 944, 946. doi: 10.2144/01314rr05.
Here, we describe a suppression PCR-based method for directional cloning of randomly primed cDNAs from small quantities of tissue. Synthesis of the first cDNA strand is conducted on oligonucleotide-coated magnetic beads. Synthesis of the second strand is accomplished using nonspecifically primed suppression PCR. This method is used to synthesize a cDNA library from zebrafish embryos at 6-9 h after fertilization. The sequencing of the clones and their use in an in situ hybridization screen to detect restricted patterns of gene transcription in zebrafish embryos showed that this method allows the rapid identification of genes that are important for development and genes that are expressed at levels undetectable by whole-mount in situ transcript hybridization. The random priming of cDNA alleviates the problems encountered in the identification of zebrafish genes from poly(dT)-primed cDNA clones caused by the long 3' UTRs frequently found in transcripts from this organism.
在此，我们描述了一种基于抑制性PCR的方法，用于从少量组织中定向克隆随机引物cDNA。第一链cDNA的合成在寡核苷酸包被的磁珠上进行。第二链的合成通过非特异性引物抑制性PCR完成。该方法用于从受精后6 - 9小时的斑马鱼胚胎合成cDNA文库。对克隆进行测序并将其用于原位杂交筛选以检测斑马鱼胚胎中基因转录的受限模式，结果表明该方法能够快速鉴定对发育重要的基因以及以整体原位转录杂交无法检测到的水平表达的基因。cDNA的随机引物设计缓解了从聚（dT）引物cDNA克隆鉴定斑马鱼基因时遇到的问题，这些问题是由该生物体转录本中经常发现的长3'UTR引起的。
