Abstract
Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80 % GC contents and a size of >300 bp. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65–85 % and sizes up to 870 bp. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.
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Acknowledgments
Some of the primers for amplification of GNAS and those of MCART1 were designed for our collaborative project with University of Lübeck, Germany and were kindly provided for this study by Dr. Christine Klein. We are grateful to Ms. Rasheeda Bashir for independently verifying this protocol with primers for TPRN. We thank Dr. Edward Wilcox for his valuable comments on the manuscript. The research was supported by Higher Education Commission, Pakistan, (Grant #836) and Fogarty International Center and National Institute on Deafness and other Communication Disorders, National Institutes of Health, USA, (R01TW007608).
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Sadaf Naz and Amara Fatima contributed equally to this study.
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Naz, S., Fatima, A. Amplification of GC-rich DNA for High-Throughput Family-Based Genetic Studies. Mol Biotechnol 53, 345–350 (2013). https://doi.org/10.1007/s12033-012-9559-y
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DOI: https://doi.org/10.1007/s12033-012-9559-y