Previously described was:

"A Novel Isothermal Nucleic Acid Amplification Resulting in a Spatially Localized Product"

Therein was claimed an exponential nucleic acid amplification resulting in a spatially localized product, and a derivation thereof unitizing a linear template was described.

Herein is described another derivation utilizing circular templates. Figure 1 exemplifies the process.

Of essence are primers which after being hybridized to the primary RCA product, are hybridized to other RCA templates (which in this example are different from the primary RCA templates). The 5' portion of these primers are designed such that they strongly associate with the primary RCA product. This can be accomplished simply by making these 5' portions sufficiently long, or by their modification. Such modifications would include incorporation of nucleotide derivatives, such as those to which intercalators are conjugated, those which constitute peptide nucleic acids (PNA), and those which are chemically reactive so as to cross-link the hybrid. "Clamping" triplex forming sequences could also be utilized.

Conceivably, the 5' portion of a primer could be replaced by any molecule which selectively binds sites on the primary RCA product. For example, the primary RCA product could comprise biotin-conjugated nucleotides which are targeted by primer-conjugated avidin.

In Figure 1 the process comprises steps, particular the removal of those primers not hybridized to the primary RCA product. It would be advantageous to have a one-step one-pot process, and such a process can be effected utilizing a latent primer such as shown in Figure 2.

For clarity, only a small section of the primary RCA product, comprising the latent primer binding site, is shown. (Note the latent primer binding sequences (red) could be contiguous).

The latent primer hybridized to this primary RCA product section comprises 3 portions. A 5' portion (pink) which stably hybridizes to the primary RCA product; a internal portion (light green) which will function to prime the secondary RCA template; and a 3' portion (pink) which also hybridizes to the primary RCA product. This third portion also has a 3 'modification preventing its extension.

Importantly, at the junction of the internal and 3' portions is one or more scissle bonds, cleaved for example by a nicking enzyme, a restriction enzyme, or by an RNase. The result of this cleavage is to activate the latent primer, which goes on to prime the secondary RCA template.

Claims

As with all my inventions this is only at the conceptual stage, and it is hoped that others who have laboratory facilities will investigate its full potential.

Addendum 4/11/05:

While perhaps known, it is worthy of mention that a simple means of amplifying the signal from any amplified nucleic acid, would be to first incorporate ligand-conjugated nucleotides (such as biotin-UTP) into the product, and then provide label-conjugated ligand-binding molecules (such as alkaline phosphatase-avidin).
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Addendum 4/14/05:

Here is a highly relevant reference, yet importantly the claimed process is a step-wise process which does not utilize the novel primers as exemplified in the Figure 2 below.

Poly-primed amplification of nucleic acid sequences Kingsmore, et al United States Patent Application 20010041340
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Addendum 4/15/05: Figure 3 represents another form of latent primer allowing for one-step isothermal exponential amplification.

Claim: A one-step spatially localized isothermal exponential nucleic acid amplification utilizing latent primers.
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