Detection Of Mercury And Single-nucleotide Polymorphism Using Rolling Circle Amplification

Nucleic acid is one of the most important biomacromolecules. Analysis of nucleic acids plays a very important role in life science. Detection of special sequence of DNA and single nucleotide polymorphism (SNP) is important in clinical diagnosis, analysis of medicament and so on. Mercury pollution pervades the globe and remains a danger to human health. Rolling circle amplification (RCA) is an isothermal nucleic acid amplification method which is simple,rapid,highly sensitive and specific. RCA has a broad application prospects for detection of nucleic acid and SNP detection. In this we paper mainly developed new methods for detection of SNP and mercury by combining RCA with fluorescence detection.Combination of high sensitivity and specificity of RCA with fluorescence resonance energy transfer (FRET) technology, a new method for homogeneous SNP detection has established. The padlock probe was designed, in which 3'and 5'terminus sequences were perfectly complementary to the mutant DNA. The padlock probe was ligated by using the mutant DNA as template to form circle probe. Then, the mutant DNA can act as a primer to initiate RCA. During the RCA, Fluorescence-labeled dGTP is incorporated into the new DNA strand produced by RCA. Upon adding the cationic conjugated polymer (CCP), strong electrostatic interactions between DNA and CCP bring them close and efficient FREF from CCP to fluorescein occurs. The wild DNA contains a mismatch base with the padlock probe. Therefore, the padlock probe can not be ligated and RCA can not take place. According to the significant difference of the fluorescent signals between mutant DNA and wild DNA, highly sensitive and specific detection of DNA can be realized. The method can accurately measure 1% mutations.The circle DNA probe containing nine T base was designed. Mercury ions can specifically connect T-T bases by formation of T-Hg2+-T structure. We design a primer sequence containing nine T bases which can interact with the circle probe by introduction of mercury ions. Then, RCA is initiated by using DNA polymerase with the replacement activity. The products of RCA are determinated by specific fluorescent dye SYBR Green I. So a new method for sensitive detection of mercury is proposed. Detection limit of mercury is 1 nmol/L.