RCAT^TM Technology Details

RCAT is a highly sensitive and efficient amplification method that has been configured in a number of formats to address a broad range of applications.

Linear RCAT (L-RCAT)

One end of a DNA probe is joined to the desired target.

The other end is hybridized to a generic DNA circle.

An enzyme catalyst is added. Starting at the probe for the circle, the enzyme catalyst makes a copy of the DNA circle, returns to the starting point and continues to copy the circle.

A single product is thus generated that is composed of copy after copy of the circle like a thread unraveling from a spool.

Within several hours RCAT produces thousands of copies of the circle as a single ribbon of DNA at a rate of 55 nucleotides per second.

The newly synthesized, long ribbon of linked-up copies of the circle that is the product of the RCAT reaction folds itself up into a tiny speck that is attached to the target. When fluorescent detector molecules are used, the result is an intensely bright signal at the location of each single target biomolecule.

Copies of the circle remain linked to the target, not dispersed in solution or distributed throughout samples.

Exponential RCAT (E-RCAT)

A second short DNA probe, identical in sequence to part of the DNA circle is added.

Upon incubation, each newly formed linear copy of the circle is recognized by this second DNA probe, which, in turn makes copies of the RCAT product.

In this way, amplification and displacement take place both around the circle and on the RCAT product.

The resultant amplified material is a so-called hyper-branched product that is produced in exponentially increasing amounts.

Multiply Primed RCAT (M-RCAT)

Oligonucleotide primers complementary to the amplification target circle are hybridized to a circular DNA template generating multiple replication forks.

Polymerase is added and RCAT proceeds by displacing the non-template strand. Product strands are rolled off the template as tandem copies of the circle.

Priming allows synthesis of both strands resulting in double-stranded product.

A cascade of priming events results in exponential (or hyper-branched) amplification.

Next: Applications- RCAT on Microarrays