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GSJ: Received June 24 2005:
http://wbabin.net/saba/saba53.htm
Nucleic Acid Amplifications utilizing Polymers or Particles Conjugated with Multiple Primers
James Saba
Herein is presented novel of means of nucleic acid amplifications, many variations of which result in a spatially localized product and are isothermal. Of essence to the invention is the use molecules (preferably linear or branched polymers) to which oligonucleotide primers are conjugated. Alternatively, the molecules can be replaced by a material support (such as nanoparticle or microarray locus) to which multiple identical oligonucleotide primers are conjugated.
Figure 1 exemplifies the invention as a modification of PCR, wherein instead of having individual primers, we utilize a branched molecule (thin lines) to which multiple identical primers have been conjugated. For convenience we will call a multiple-primer-conjugated molecule or material support a 'polyprimer'.
We start by hybridizing a template with one of two different polyprimers. Subsequent to polymerase elongation of the hybridized polyprimer, the duplex product is separated with heat. One product thereof, the unaltered target can be recycled with a new or partially utilized polyprimer. The other product from denaturation is hybridized with the second polyprimer, which is subsequently elongated. The steps of denaturation, hybridization new or partially used polyprimers, and elongation are repeated as desired. Notice that under appropriate condition there is considerable hybridization-based crosslinking, and spatial localization of product.
An important PCR-like variation of Figure 1 is where multiple copies of one PCR primer is affixed to a support (such as microarray locus), and the second PCR primer is in polyprimer form.
Polyprimers also have utility in isothermal nucleic acid amplifications as shown in Figures 2 and 3.
Figure 2 is a simple depiction of the basic processes involved in one form of isothermal amplification which involves primer invasion and strand displacement (1-3). We start by hybridizing a ssDNA template to one primer of the polyprimer, followed by polymerase extension. Subsequently there is invasion by an adjacent primer of the polyprimer, and concurrent with its extension the first polymerization product is displaced. With two more cycles of primer invasion, extension and displacement we finish with the polyprimer wherein each primer is extended.
Notice the template could be affixed to a molecule such as an antibody, or to a support such as a bead or microarray locus.
While the degree of amplification from the process in Figure 2 is small, derivations of this basic process can lead to exponential amplifications, as depicted in Figure 3.
In the above Figure 3, we make use of two different polyprimers, and a support-affixed template. Importantly, the sequence at 5' portion (red) of the template is the same as that of the secondary primers (red) on the polyprimer. This initial processes in Figure 3 are essentially the same as those of Figure 2. Distinctly however, the displaced product itself functions as a template for the secondary polyprimer (red).
Notice that the displaced product could function is other ways, such as prime a rolling circle amplification template. Furthermore, it is conceivable that with the use of stem-loop templates, only one polyprimer would be required.
Variations of the above invention are possible, and following provisional claims attempt to encompass these variations.
Claims
2) A nucleic acid amplification which utilizes two different soluble multiple-primer-conjugated molecules.
3) The amplification of claim 1 or 2 wherein the primers conjugated to a molecule are identical.
4) The amplification of claim 1, 2 or 3, wherein the molecule is a linear or branched polymer.
5) The amplification of claim 4, wherein the polymer is PEG.
6) A nucleic acid amplification which utilizes a multiple-primer-conjugated particle, and wherein the primers conjugated to a particle are identical.
7) A nucleic acid amplification which utilizes two different multiple-primer-conjugated particles, and wherein the primers conjugated to a particle are identical.
8) The amplification of claim 6 or 7, wherein the particle is less than 10 micrometers.
9) The amplification of claim 6 or 7, wherein the particle is less than 1 micrometer.
10) The amplification of claim 6 or 7, wherein the particle is less than 100 nanometers.
11) The amplification of any above claim, wherein the amplification is isothermal.
12) The amplification of any above claim, wherein the amplification is exponential.
13) The amplification of any above claim, which is initiated by the multiple-primer-conjugated molecule or particle hybridizing to a polynucleotide target which is affixed to a molecule or material support.
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