A Novel Isothermal Nucleic Acid Amplification

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GSJ:Received January 14, 2005: http://wbabin.net/saba/saba17.htm

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

James Saba

While there are numerous nucleic acid amplification techniques, few thereof result in a spatially localized product. Such amplifications are particularly valuable when the initiating primer is affixed to a support such as a microarray chip or bead, or to a molecular probe such as an antibody. The currently most attractive means of forming a spatially localized nucleic acid amplification product is rolling circle amplification (1-3).

Herein is disclosed another isothermal nucleic acid amplification which results is a spatially localized product, which due to the lack of topological constraints inherent in rolling circle amplification, may have greatest utility. Distinctly from rolling circle amplification, herein the nucleic acid hybridized to the affixed primer is linear. Subsequently, successive linear polynucleotides are added to form a substantially linear double stranded product.

Figure 1 depicts a preferred embodiment of this process. Initially the affixed primer is hybridized to a specially designed linear single-stranded polynucleotide, the length of which may be considerably larger than that shown. Each end of this linear polynucleotide comprises the same sequence element, yet in this example the 3' end comprises a modification which enhances it invasive hybridization capacity (4-6). Herein the modification is a peptide nucleic acid sequence (hatched section). Another example of such a modification would be a peptide which enhances stand invasion. It is to be appreciated, in the broadest sense of the invention, such a 3' modification is not required.

Subsequent to hybridization the primer is extended by nucleotide polymerization to form a double-stranded nucleic acid product. The terminus of this product, distal the support, is then invaded by another single-stranded linear polynucleotide, which in this example its identical to that hybridized to the initiating primer. The first cycle is completed by polymerase extension along the newly incorporated polynucleotide.

Figure 2 depicts a second preferred embodiment of the process. Therein, rather than strand invasion, a 5' terminal modification(s) of the linear polynucleotide renders an internucleotide bond(s) therein scissle subsequent to duplex formation (7,8). Examples of 5' terminal modifications would include ribonucleotides cleaved by RNaseH, or nickable sequences. It may be desirable to configure the linear polynucleotide's 5' terminal sequence such that it does not readily hybridize, such a designing a hairpin structure.

Finally it is to be appreciated that the initiating primer need not be support affixed, and that amplifications products may be subjected to processing such as further types of amplifications, and/or various labeling schemes (9).

Addendum 1/19/05
Claims

1) A nucleic acid amplification process comprising adding successive nucleic acids.

2) The amplification process of claim 1, wherein the nucleic acid is a linear polynucleotide (optionally have secondary structure).

3) The amplification process of claim 1, wherein the linear polynucleotide is modified at its 3' end such that it invades the terminus of, and hybridizes to one of the strands of double stranded nucleic acid.

4) The amplification of claim 3, wherein the 3' modification is a peptide nucleic acid sequence.

5) The amplification of claim 3, wherein the 3' modification is a peptide.

6) The amplification process of any of claims 1-6, wherein the amplification results in a substantially linear double stranded product.

Addendum 1/31/05

Exponential, Spatially Localized Isothermal Nucleic Acid Amplification

Recently a new type of nucleic acid amplification resulting in a spatially localized product was disclosed.

The self explanatory figure below is a modification of that process involving successive additions of templates, wherein the product is formed in an exponential rather than linear fashion. The essence of the invention is the use of branched latent primer templates.

Such an amplification is anticipated to be exceptionally useful, particular in the field of microarrays.

It appears likely other such derivations are possible, and all such derivations are to be encompassed by the following claims

Claims

1) An exponential nucleic acid amplification resulting in a spatially localized product.

2) An isothermal exponential nucleic acid amplification resulting in a spatially localized product.

References
1) Detection of target nucleic acids and proteins by amplification of circularizable probes (review).
Zhang, et al Expert Rev Mol Diagn. 2003 Mar;3(2):237-48

2) Multiplexed protein profiling on microarrays by rolling-circle amplification.
Schweitzer, et al Nat Biotechnol. 2002 Apr;20(4):359-65

3) Multiplex detection of hotspot mutations by rolling circle-enabled universal microarrays.
Ladner, et al Lab Invest. 2001 Aug;81(8):1079-86

4) Strand invasion by mixed base PNAs and a PNA-peptide chimera.
Zhang, et al Nucleic Acids Res. 2000 Sep 1;28(17):3332-8

5) Enhanced strand invasion by peptide nucleic acid-peptide conjugates.
Kaihatsu, et al Biochemistry. 2002 Sep 17;41(37):11118-25

6) Extending recognition by peptide nucleic acids (PNAs): binding to duplex DNA and inhibition of transcription by tail-clamp PNA-peptide conjugates.
Kaihatsu, et al Biochemistry. 2003 Dec 2;42(47):13996-4003

7) Nucleic acid compositions with scissile linkage useful for detecting nucleic acid sequences.
Duck,et al United States Patent 4,876,187 Oct 24, 1989

8) Method for amplifying nucleic acid sequences by strand displacement using DNA/RNA chimeric primers.
Cleuziat, et al United States Patent 5,824,517 Oct 20, 1998

9) Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets.
Kuhn, et al J Am Chem Soc. 2002 Feb 13;124(6):1097-103M

Addendum 6/29/05

If the stability of a parallel nucleic acid duplex is sufficient, and a strand thereof can be displaced by a polymerase, then an amplification via successive additions of a nucleic acid as depicted in the following figure is possible.