In previous papers (1,2) novel latent primers were described which have the capacity to specifically hybridize to a target sequence and prime an amplification reaction.

Herein is also described novel means of detecting, characterizing (such as for SNPs) and/or quantifying specific sequences, particularly those affixed to a support such as found in in situ hybridization.

The essence of the invention, as shown in Figure 1, is the formation of a primer via nicking of a target sequence which has been hybridized to an amplification template, particular a circular RCA template.

In this example we start with a cell or spot of denatured DNA which has a sequence (red) which we wish to identify and/or quantify.

We next hybridize the red target sequence with a sequence present on an amplification template, in this case a rolling circle template.

We next provide a nicking agent to nick the hybridized target sequence so as to form a primer. Nicking can be done for example with nicking enzymes, restriction enzymes, or mismatch enzymes. If utilizing restriction enzymes the RCA template sequence complementary to the nicking site is designed to resist cleavage (such as by a phosphorothioate linkage).

Subsequently the newly formed primer is extended with nucleotides. Labeling can be achieved in various ways, herein labeled nucleotides are utilized.

An interesting means of reducing background primer extension is to design an amplification template of only three nucleotides, and only provide these three nucleotides in the amplification reaction. With one nucleotide missing, primer extension not directed by the amplification template would quickly come to halt.

'The process outlined in Figure 1 appears to be simpler than either that described by Christian, et al (4) or Zhong, et al (5).

Finally, note that the linear templates as described in (3) could be advantageously utilized, particularly in in situ hybridization, and that denaturization of target sequences would not necessarily be needed.

Provisional claims:

Addendum 4/18/05 :

Of course the target of this process can be any polynucleotide, including those which are soluble and those produced via an amplification.

Claim 1: A process comprising:

Addendum 4/27/05:

Almost certainly known, but worth mention would be to utilize the primers described in the prior paper "Spatially Localized Exponential Rolling Circle Amplification (RCA)" directed to support bound, naturally occuring nucleic acids as in Figure 1 above.

Particularly interestingly, there are known nucleotide derivatives (1,2) which when incorporated into polynucleotides. covalent couple to a target upon hybridization of this polynucleotide to the target. That is hybridization triggers covalent coupling to the target.

1) Rapid and efficient hybridization-triggered crosslinking within a DNA duplex by an oligodeoxyribonucleotide bearing a conjugated cyclopropapyrroloindole.
Lukhtanov, et al Nucleic Acids Res. 1996 Feb 15;24(4):683-7.

2)Hybridization triggered cross-linking of deoxyoligonucleotides.
Webb, et al Nucleic Acids Res. 1986 Oct 10;14(19):7661-74.
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Addendum 5/11/05

As as alternative to the process shown in Figure 1, target DNA could be first cleaved with one or more restriction enzymes, optionally subjected to exonuclease digestion as described by Christian, et al (4), and then a 3' end sequence could be hybridized to a RCA template.