Universal method facilitating the amplification of extremely GC-rich DNA fragments from genomic DNA.
Polymerase chain reaction (PCR) is a basic technique with wide applications in molecular biology. Despite the development of different methods with various modifications, the amplification of GC-rich DNA fragments is frequently troublesome due to the formation of complex secondary structure and poor denaturation. Given the fact that GC-rich genes are closely related to transcriptional regulation, transcriptional silencing, and disease progression, we developed a PCR method combining a stepwise procedure and a mixture of additives in the present work. Our study demonstrated that the PCR method could successfully amplify targeted DNA fragments up to 1.2 Kb with GC content as high as 83.5% from different species. Compared to all currently available methods, our work showed satisfactory, adaptable, fast and efficient (SAFE) results on the amplification of GC-rich targets, which provides a versatile and valuable tool for the diagnosis of genetic disorders and for the study of functions and regulations of various genes.
Wei
Maochen
M
Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, People's Republic of China.
Deng
Jing
J
Feng
Kun
K
Yu
Boyang
B
Chen
Yijun
Y
D000818:Animals; D006224:Cricetinae; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D020862:GC Rich Sequence; D016678:Genome; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004247:DNA