Klein V, Piontek K, Brass N, Subke F, Zang K D, Meese E
Department of Human Genetics, University of Saarland, Homburg/Saar, Germany.
Genet Anal Tech Appl. 1993 Feb;10(1):6-9. doi: 10.1016/1050-3862(93)90018-e.
Recently, methods have been developed for the isolation of expressed sequences from particular human chromosomes. Using Alu consensus sequences as primers, cDNA synthesis has been initiated from interspecies hybrid cell lines that contain single human chromosomes. Alu consensus sequences have also been utilized to amplify human genomic sequences via polymerase chain reaction (PCR). Here, we describe the use of Alu-PCR to isolate expressed sequences from human chromosomes selectively. Heteronuclear (hn) RNA is transcribed into cDNA by using poly-(dT)15 primer sequences. Subsequently, human specific cDNA sequences are amplified by Alu-PCR and cloned into pBluescript. To verify the chromosomal assignment, cloned PCR products are sequenced, converted into STS markers, and tested on a different somatic hybrid that contains human chromosome 22. The method provides a fast, reliable way to identify expressed sequence tagged sites from selected human chromosomes.
最近，已经开发出了从特定人类染色体中分离表达序列的方法。以Alu共有序列作为引物，从含有单条人类染色体的种间杂交细胞系中起始cDNA合成。Alu共有序列也已被用于通过聚合酶链反应（PCR）扩增人类基因组序列。在此，我们描述使用Alu-PCR从人类染色体中选择性分离表达序列的方法。通过使用聚（dT）15引物序列将异核（hn）RNA转录成cDNA。随后，通过Alu-PCR扩增人类特异性cDNA序列并克隆到pBluescript中。为了验证染色体定位，对克隆的PCR产物进行测序，转化为STS标记，并在含有人类22号染色体的不同体细胞杂种上进行测试。该方法提供了一种从选定的人类染色体中鉴定表达序列标签位点的快速、可靠的方法。
