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Successful PCR amplification and subcloning of a GC-rich DNA fragment

Petra Majlingov, Paolo Paroncini, Silvia Svegliati, Michele Luchetti, and Armando Gabrielli
Laboratorio di Medicina Molecolare, Istituto di Clinica Medica, Polo Didattico Scientifico, Ancona, Italy

The formation of robust secondary structures that resist denaturation makes GC-rich sequences difficult to amplify by PCR. This article demonstrates how a "difficult" GC-rich sequence was successfully amplified using HotStarTaq DNA Polymerase and Q-Solution. The PCR product was then ligated into the pDrive Cloning Vector, which was used to transform QIAGEN EZ Competent Cells with high efficiency.

Our group has been working for many years on the regulation of expression of the myb family of transcription factors (13). The myb gene family is made up of three genes, c-myb, A-myb, and B-myb, whose pattern of expression is controlled by complex mechanisms. For example, c-myb expression is regulated by a mechanism of transcriptional block in the first intron and alternative splicing.

In order to further investigate the role of the c-myb intron in expression, we attempted to amplify the Myb-INT gene fragment. Myb-INT is a 4500 bp DNA fragment from the first c-myb intron, and has a GC content of about 60%. Sequences that contain a high ratio of guanosine and cytidine base pairs (GC-rich sequences) can form robust secondary structures that resist denaturation and prevent annealing of PCR primers.

PCR was performed using HotStarTaq DNA Polymerase and 4 other commercially available DNA polymerases. Of the 5 polymerases tested, only HotStarTaq DNA Polymerase (in conjunction with Q-Sol
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