通过凝胶内竞争性重退火分离多态性mRNA（cDNA）

Isolation of polymorphic mRNAs (cDNAs) by in-gel competitive reassociation.

作者信息

Gotoh Koshichi, Oishi Michio

机构信息

Human Gene 2, Kazusa DNA Research Institute, 2-6-7, Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.

出版信息

Genomics. 2004 Aug;84(2):435-40. doi: 10.1016/j.ygeno.2004.03.002.

DOI:10.1016/j.ygeno.2004.03.002

PMID:15234006

Abstract

In-gel competitive reassociation (IGCR) is a method for differential subtraction of polymorphic (RFLP) DNA fragments between two DNA samples of interest without probes or specific sequence information. Previously IGCR was used to enrich and isolate polymorphic genomic DNA fragments from mouse and human genomic DNA. We have modified the original IGCR procedures specifically for the isolation of polymorphic mRNAs in the form of cDNAs. Here we demonstrate that polymorphic mRNAs (cDNAs) between BALB/c and C57BL/6J mice that result from alterations at restriction sites and insertions-deletions are isolated with high efficiency by the IGCR procedure. A high proportion of the cDNAs was enriched by IGCR and 80% of the enriched clones were found to be actual RFLP fragments, indicating that the procedure is also applicable to direct screening for polymorphic mRNAs.

摘要

凝胶内竞争性复性（IGCR）是一种无需探针或特定序列信息，就能对两个感兴趣的DNA样本之间的多态性（RFLP）DNA片段进行差异消减的方法。此前，IGCR被用于从小鼠和人类基因组DNA中富集和分离多态性基因组DNA片段。我们对原始的IGCR程序进行了改进，专门用于以cDNA形式分离多态性mRNA。在此，我们证明通过IGCR程序可高效分离BALB/c和C57BL/6J小鼠之间由限制性位点改变和插入-缺失导致的多态性mRNA（cDNA）。大量的cDNA通过IGCR得到富集，并且发现80%的富集克隆是实际的RFLP片段，这表明该程序也适用于直接筛选多态性mRNA。