Improvement of RCA initiation on genomic DNA

In this thesis, we tried to improve the initiation of Rolling Circle Amplification (RCA) on genomic DNA using an approach known as Single Primer Extension (SPE). Usually, a synthetic DNA target provides a high signal on RCA after padlock probe hybridisation due to its short 3’ end. However, unfragmented genomic DNA provides low efficiency for RCA initiation due to its long 3’ end. During SPE, the genomic DNA is used as a template and a biotinylated primer amplifies this target to generate a product of limited length. This amplified product is then captured on to streptavidin-coated magnetic beads. Padlocks specific to the target DNA is then added. They hybridise to the target and closed into circles with the help of a Tth ligase enzyme. Replication of the circularised padlock takes place by a method known as RCA. The Rolling circle products (RCPs) are further amplified with the help of Circle-to-Circle Amplification (C2CA). The RCPS are hybridised with a fluorophore tagged Detection Oligonucleotide (DO) and readout as images containing individual blobs, using a microfluidic system placed on a confocal microscope. The efficiency of SPE was compared with a target which was precoupled to the streptavidin-coated magnetic beads prior to padlock hybridisation and ligation. It was seen that a single cycle of RCA was as good as its pre-coupled target control but it was not better than the Standard C2CA genomic DNA control even after changing several factors of SPE. Cycling of SPE always provided a high signal, close to the Std-C2CA synthetic target control. Also a Pre-PCR amplification was tested in this project but did not show any success during the few experiments conducted. Therefore, cycling of SPE could be used as option to test the padlocks on genomic DNA.