使用全染色体直接筛选互补DNA

Direct selection of cDNAs using whole chromosomes.

作者信息

Rouquier S, Trask B J, Taviaux S, van den Engh G, Diriong S, Lennon G G, Giorgi D

机构信息

Centre de Recherche de Biochimie Macromoléculaire, CNRS UPR 9008, Montpellier, France.

出版信息

Nucleic Acids Res. 1995 Nov 11;23(21):4415-20. doi: 10.1093/nar/23.21.4415.

DOI:10.1093/nar/23.21.4415

PMID:7501464

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC307398/

Abstract

We have developed a method for direct selection of cDNAs using whole chromosomes as target DNA. Double-strand cDNAs were synthesized from human fetal brain polyadenylated mRNAs. Flow-sorted chromosomes 17 and 19 were amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and used to capture ds cDNAs by an improved magnetic bead capture protocol. To demonstrate the capabilities of this method, the selected cDNAs were used as probes in FISH experiments. The selected cDNA populations specifically painted chromosomes 17 or 19 on metaphase spreads. These results demonstrate that it is possible to do chromosome painting using cDNA probes and that this method is a means to rapidly select expressed sequences encoded by any portion of the genome.

摘要

我们已经开发出一种以整条染色体作为靶DNA直接筛选cDNA的方法。双链cDNA由人胎儿脑多聚腺苷酸化mRNA合成。通过简并寡核苷酸引物聚合酶链反应（DOP-PCR）扩增流式分选的17号和19号染色体，并采用改进的磁珠捕获方案捕获双链cDNA。为了证明该方法的能力，将筛选出的cDNA用作荧光原位杂交（FISH）实验的探针。筛选出的cDNA群体在中期染色体铺展上特异性地描绘出17号或19号染色体。这些结果表明，使用cDNA探针进行染色体描绘是可行的，并且该方法是快速筛选基因组任何部分编码的表达序列的一种手段。