Development of a PCR-free direct genomic DNA detection assay using rolling circle amplification.

摘要

As the information carrier in nature, DNA also represents a unique "fingerprint" for each living organism. In the biodetection, biosecurity, and diagnostic fields, DNA detection assays have been developed to detect the presence of different organisms using a variety of methods. Polymerase chain reaction (PCR) is considered the gold standard for DNA detection but does have drawbacks. The development of a comparable assay to PCR without necessitating the purchasing of additional PCR equipment is therefore quite desirable, especially in developing countries.;Rolling Circle Amplification (RCA) is a useful technique for amplifying the presence of a single target DNA sequence. A polymerase extends the original target by "rolling" a circular RCA primer sequence; the extension consists of many tandem repeated copies of this primer. A sandwich capture assay was initially developed in which these RCA-generated products were measured in real-time during extension. Following heat extraction to denature the reporter probe, extension was performed in a well plate, and monitored in real-time with the use of SybrGreenII. Over one hour, 10 pM of target gave a measurable signal over background.;To develop the assay further, the sandwich capture concept was applied to large polymer beads. Here, RCA was done directly on immobilized targets on bead surfaces, and the RCA products were labeled with a fluorescent complementary probe. These amplified target molecules appear as "dots" and can be easily counted. The sensitivity was drastically enhanced; detection of 1 amol of target sequences was obtained.;Detection of double-stranded DNA targets was attempted by using padlock and capture probes as "blocking" sequences which, after a denaturing protocol was performed, prevent the re-hybridization of the double stranded target via steric hindrance. After optimization, the assay was able to detect dsDNA targets at concentrations of 425 aM. As an application, the direct detection of genomic DNA extracted from Mycobacterium tuberculosis was attempted. After double restriction enzyme digestion, DNA was detected in bacterial concentrations as low as 103 cfu/mL, comparable to PCR sensitivity. The specificity of the assay was confirmed when no significant signal was obtained for two separate bacterial genomic DNA samples.