Assay Of Nucleic Acids Based On Rolling Circle Amplification

By mimicking the rolling circle replication in some pathogenic microorganisms, a technology named as rolling circle amplification(RCA) is developed for isothermal amplification of nucleic acids. With the advantages of simplicity and high efficiency, RCA attracts more and more attentions in life science since its first report. Traditional RCA consists of three critical components: a circular template, a primer and a DNA polymerase. In a RCA reaction, the primer is extended to a linear single stranded DNA with short tandem repeats which are complementary with the circle DNA template. This is the first generation of RCA, also referred to as linear rolling circle amplification(LRCA). In recent years, new generations of RCA have also been developed, such as hyperbranched rolling circle amplification(HRCA), netlike rolling circle amplification(NRCA), etc. In this study, we focused on the combination of RCA with nanomaterials or some other nucleic acids techniques and thus to investigate the novel application of RCA for the assay of nucleic acids. In the first chapter, we developed a nanomaterials-assisted RCA for the assay of single nucleotide polymorphism(SNP) of mi RNA. In the second chapter, we developed a modified RCA for the assay of the DNA break events. Results show that the above two approaches have prospect for further development and application.1. Reduced graphene oxide-assisted rolling circle amplification for the assay of single nucleotide polymorphism of Mi RNABecause a single mi RNA may regulate the expression of hundreds of genes, mi RNA SNPs can result in a high incidence of extensive cellular transformation and various diseases. Therefore, the assay of mi RNA SNPs has important medical values. In this chapter, we developed a high specific nucleic acids amplification technique named as reduced graphene oxide assisted rolling circle amplification(r GO-RCA). In our system, reduced graphene oxide was used as an auxiliary material to improve the specificity of RCA and consequently assay mi RNA SNPs. Agarose gel electrophoresis was adopted to characterize the conditions optimization. Fourier transformed infraredspectrum(FITR) was used to study the mechanisms. Atomic force microscopy(AFM) was used to characterize the amplified products. At the end, fluorescence quantitative PCR and fluorescence spectrum were adopted for the quantitative determination of mi RNA SNPs. Results show that the difference of the signal of a mi RNA SNP even reaches 100 fold, which is over 10 times larger than most current methodologies. Detection of a G-U polymorphism of mi R-125 a, and an A-G polymorphism of let-7a was thereby achieved. In addition to the ultrahigh specificity, the sensitivity as well as the usability has also been demonstrated to be favorable, making this r GO-RCA strategy a promising alternative for profiling mi RNA SNPs in lab or clinic.2. Terminal blocked rolling rircle amplification for the assay of DNA break eventsDNA break is one of the major kinds of DNA damage. Sensitive assay of DNA break events is the prerequisite for the study of damage inducements and their mechanisms. In this chapter, a novel RCA technique termed as terminal blocked rolling circle amplification(TB-RCA) is proposed for the assay of DNA break events. Briefly, a terminal blocked primer was introduced into RCA system. In the absence of DNA break events, the primer kept blocked and the process of RCA is inhibited. Otherwise, while the primer was cleaved(a break state), the blocking terminal sequence was cut off; and the primer was activated, making the RCA be launched. As a consequence, we are able to sensitively screen the DNA break events. Agarose gel electrophoresis was used for qualitative assay and conditions optimization. And, fluorescence spectrum was adopted for quantitative assay. Fenton reaction and endonucleases were adopted as model inducements for DNA break to study the practicability. Results show that sensitive assay of the DNA break events can be achieved by using the TB-RCA. In comparison with most current available methods, our method has advantages of simplicity, high sensitivity and versatility. This method also provides a potential path for the assay of DNA break events in vitro.