Rolling circle amplification technology
Rolling circle amplification technology (RCAT), a proprietary amplification process developed by Molecular Staging Inc, has significant advantages in terms of sensitivity, multiplexing, dynamic range and scalability. The steps of this procedure are:
1. A short DNA probe anneals to a target DNA of interest, such as the DNA of a pathogenic organism or a human gene containing a deleterious mutation. The probe then acts as a primer for a Rolling Circle Amplification reaction (see 2).
2. The free end of the probe anneals to a small circular DNA template. A DNA polymerase (white oval) is added to extend the primer.
3. The DNA polymerase extends the primer continuously around the circular DNA template generating a long DNA product that consists of many repeated copies of the circle.
4. By the end of the reaction, the polymerase generates many thousands of copies of the circular template, with the chain of copies tethered to the original target DNA. This allows for spatial resolution of target and rapid amplification of the signal. The use of forward and reverse primers can change the above linear amplification reaction into an exponential mode that can generate up to 1012 copies in 1 hour.
RCAT can achieve the following:
1. Detect single target molecules or 'analytes'.
2. Amplify signals from proteins as well as DNA and RNA.
3. Pinpoint the location of molecules that have been amplified on a solid surface (in situ analysis/ biochips) since, unlike PCR, the amplified product remains attached to the target molecule.
4. Measure many different targets simultaneously.
5. Improve the ease and accuracy of quantification.
6. Simplify haplotype identification through phasing.
7. Increase sensitivity with up to 1012 -fold amplification in one hour.
8. Amplify DNA templates that vary in length from 1 base pair to over 100 kilobases.
9. Obviate the need for the time-consuming and expensive steps of thermal cycling currently.
10. Analyze targets in solution or solid phase.
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