Cell-free protein synthesis using multiply-primed rolling circle amplification products.

Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers.

The predominant method for DNA cloning is by propagation in biological hosts, but this method has limitations because certain sequences are difficult to clone using any combination of available hosts or vectors. Recently, multiply-primed rolling circle amplification (MPRCA) has been applied to overcome the problems of the DNA cloning via host cells. However, when MPRCA is used to amplify from m...

Amplification of Human papillomavirus genome using multiply primed Rolling- Circle Amplification

A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification.

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes,...

In situ detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS

BACKGROUND In situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling. Whilst short probes have several advantages over long probes (e.g. higher resolution and specificity) they carry fewer labels per molecule and therefore require higher amplification for detection. Furthermore, short probes relying only on hybrid...

Cell-free cloning using 29 DNA polymerase

We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5–7 kb in size by >109-fold, using 29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using 29 DNA polymerase is ‘‘background’’ DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while k...