Study On Methods Of PCR Amplification In High GC Content Human DNA

The polymerase chain reaction(PCR) technology has been developed for over 25years since its inception in 1985,and with its continuous development,it has become an indispensable experimental technology in molecular biology study.However,it is inadequate in practical applications now,especially in the high GC content of PCR amplification.Researchers have made much effort on how to conveniently amplify GC-rich sequences with a namelist of PCR additives,such as betaine,dimethyl sulfoxide,glycerol, polyethylene glycol.Novel thermal cycling patterns such as 'slowdown PCR' can also work efficiently for GC-rich DNA amplification.However,some of the measures are invalid,in this case we focus on high-GC fragment amplification method for further research.In this paper,we found the high-GC fragment database about human genomic chromosome,and based on this study we research the size,length and position distribution of high GC fragment in the human genomic DNA.At the same time,we randomly selected 98 GC-rich sequences(GC content from 60%to 80%and length from 700 to 800bp) in our research to study the effect of organic solvents,betaine,restriction endonuclease and nano-materials in high-GC amplification.In this research,it was found that the high-GC fragment mainly concentrated on the 1 and 16 chromosome while few on the 18th chromosome.The method of using restriction enzymes cutting genomic DNA to increase PCR efficiency shows a weak effect in most high GC amplification.Only a few sequences(GC content 61%and 76.7%) can be amplified while together with some non-specific product.We found that Betaine also presents a positive result in the high GC amplification but not all our high-GC collection.Further we have come to conclusion that 1,2-propanediol and ethylene glycol have a specific efficient on the high GC amplification,and slightly better than betaine.Finally,we found that some nano-materials mixing with 1,2-propanediol and ethylene glycol adding to PCR reaction system can be specifically efficient in enhancing the amplified production of high GC content human genomic DNA.