Herein is described a novel means of nucleic acid amplification utilizing rolling circle amplification (RCA).

The essence of the invention is sketched out in Figures 1 & 2.

In Figure 1 we start with a rolling circle template which has been acted on by a polymerase to produce a RCA product. For simplicity the displaced RCA product is shown as only two rounds of amplification. The rolling circle template has sequences within it (blue portions) which when duplexed (by green portions) form restriction sites.

In the first step, multiple identical nonpriming oligonucleotides hybridize with this RCA product to produce restriction enzyme sites, which are subsequently cleaved as shown in the next step.

Subsequent to inactivating the restriction enzyme, the cleaved RCA products are then hybridized with new circles. These hybrids then function to form RCA products identical to that initially used.

For clarity the process in Figure 1 is accomplished in steps, with multiple reactions and reagent additions. However, it is important to recognize that the process can be made to be continuous and isothermal utilizing the appropriate reagents and conditions.

For example, note that the restriction enzyme cleaves the primary rolling circle template and nonpriming oligonucleotides, and would do so to subsequent rolling circles if not inactivated. However this could be avoided by utilizing modified templates and oligonucleotides. Another means of avoiding this would be to utilize enzymes which selectively nick only sequences in the RCA product.

Notice that the nonpriming oligonucleotides are not strictly required. For example, utilizing appropriate conditions and reagents, the RCA product could be cleaved prior to being displaced from the rolling circle template. Another alternative to utilizing nonpriming oligonucleotides would be to have excess rolling circle templates, which could bind to an RCA product to form restriction enzyme sites. Yet another alternative is to design the RCA product such that it forms hairpins cleavable by restriction enzymes.

Notice that rolling circular templates with different sequence could be utilized for primary and subsequent RCAs.

Figure 2 is a modification wherein new RCA primers are derived from cycling probes. Herein a "cycling probe" represents any oligonucleotide which having bound to a target sequence, is subsequently cleaved and liberated from the target, such as described in references 2-5. Notice the cycling probes comprise a 5' 'tail' sequence, which while not hybridizing to the target, facilitates the hybridization of the 5' probe cleavage fragment to a rolling circle template. Dramatically, upon polymerase extension of these hybridized probe fragments to new rolling circle templates, RCA effects formation of multiple new RCA products which are now the targets for new cycling probes.

References

Addendum: (1/15/05)

It is to be appreciated that these novel probes can be arrayed on a support or conjugated to molecular probes, so as to detect or sequence targets, as shown in Figure 4.

Addendum: 1/19/05
Claims

Addendum,4/18/05:

A means of forming a primer via invader technology, via flap endonuclease was disclosed. Figure 1 is a reproduction from that article.

It has now been recognized that such latent primers would provide an excellent means of enhancing nucleic acid amplifications. Particularly, consider that the target is a sequence within a rolling circle amplification (RCA) product, and the activated primer goes on to prime another RCA template.

Note that the 3' termini of the probes need be caped to prevent extension. Of course the target could be a cellular nucleic acid or an amplification product.
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Addendum 4/19/05:

Someone from Europe has kindly pointed out a highly relevant reference,

Addendum 4/24/05

Notice the utility incorporating ribonucleotides for sites of cleavage either while duplexed (RNase H) or after displacement (single-stranded dependent RNase).

Also consider a rolling circle amplification template composed of the natural nucleotides, one of which is stratigically positioned. However, concerning the NTPs provided, one is a ribonucleoside complementary to the strategically positioned nucleotide in the template. The incorporated ribonucleotide is then the target of a ribonuclease. Finally, notice that a DNA/RNA polymerase could concievably be replaced by a carefully composed combination of DNA polymerase and RNA polymerase.
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