Amplification of GC-rich ADAMTS-2 and URG4/URGCP promoter regions with optimized combination of PCR enhancers

Authors : MELTEM ALPER, ESRA TOKAY, FERAY KÖÇKAR

Pages : 196-205

View : 25 | Download : 11

Publication Date : 2016-12-01

Article Type : Research Paper

Abstract :PCR is an effective and widely used method for the amplification of target DNA fragments in vitro. The templates that have high guanine-cytosine insert ignore into journalissuearticles values(GC); content, complex secondary structures, or short tandem repeats are difficult to amplify by the conventional PCR methods. Human genome regulatory regions such as promoters are rich in terms of GC base compositions. To amplify these regions some special modifications or additives are required. Using different PCR strategies such as hot-start/touchdown PCR or the utilization of various additives into the reaction mixture such as organic molecules can improve PCR yield. In the present study we optimized the PCR conditions for the amplification of the human ADAMTS-2 gene promoter region featuring an extremely high GC content and a secondary structure. We show that a combination of three additives, betaine, dimethyl sulfoxide, and 7-deaza GTP, is essential to obtain a correct PCR amplicon. To demonstrate whether the optimized PCR condition was applicable to other GC-enriched regions, a similar procedure was repeated for the amplification of the human URG4/URGCP gene promoter.
Keywords : GC rich PCR, PCR enhancers, promoter amplifications, robust PCR