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GSJ:Received January 18, 2005:
http://wbabin.net/saba/saba18.htm
DNA Amplifications Utilizing Unnatural Base-Pairs and a DNA/RNA Polymerase
James Saba
Several amplifications utilize nicking to produce a 3' hydroxyl which is subsequently extended. Unfortunately, in general these nicking enzymes recognize a not uncommon sequence.
Herein is proposed a new kind of amplification which obviates the need for nicking enzymes, and whose novelty and essence includes at least one of the following:
2) utilizing at least one unnatural base pairing nucleotide (3-6) in a template, primer, and/or primer precursor.
Figure 1 shows one of many examples of such an amplification. Therein we start with a single stranded circular template into which a sequence of one or more unnatural base-pairing deoxynucleotides is incorporated. Next we include both natural deoxynucleotide triphosphates (dNTPs) and unnaturally base-pairing ribonucleotide triphosphates (ØTPs) combined with a polymerase than can synthesize both DNA and RNA (DNA/RNA polymerase). After this DNA/RNA polymerase progresses to the dØ(n) by incorporation of dNTPs, it next incorporates complementary unnaturally base-pairing ribonucleotide triphosphates (ØTPs) as directed by the template dØ(n). Subsequent steps are self evident.
Figure 2 shows the use of a primer comprising unnaturally base-pairing nucleotides. Again the process should be self evident in light of Figure 1. Particularly note the second step, which utilizes an "insertion" primer comprising a modification (in this case a peptide nucleic acid sequence) which enhances it binding without the need for denaturation.
Many other processes, amplifications and other, utilizing such novel reagents are conceivable. For example polynucleotide sequences could be detected, or one or more nucleotides thereof could be sequenced utilizing cycling probes (primer precursors), or cyclizable (ligatable) linear probes; either being comprised of one one or more unnatural base-pairing nucleotides and perhaps a mixture of naturally base-pairing nucleotides.
As far as I'm aware, this is the first example of a polynucleotide comprising both deoxyribonucleotides and ribonucleotides, wherein at least one nucleotide is an unnaturally base-pairing nucleotide.
Furthermore, amplifications as in Figure 1 and 2 appear to be the first wherein a DNA/RNA polymerase is utilized, particular where unnaturally base-pairing nucleotides are involved.
Thus to adequately cover the present invention and modifications thereof the following provisional claims are made.
Claims
2) A nucleic acid amplification utilizing unnaturally base-pairing nucleotides.
3) A nucleic acid amplification utilizing a DNA/RNA polymerase, and a mixture of deoxyribonucleotide triphosphates and ribonucleotide triphosphates wherein at least one nucleotide is an unnaturally base-pairing nucleotide.
4) A nucleic acid amplification utilizing a template comprising one or more unnaturally base-pairing nucleotides.
5) A nucleic acid amplification utilizing a primer or primer precursor comprising one or more unnaturally base-pairing nucleotides.
6) The amplification process of any of claims 1-5, involving strand displacement.
7) The amplification process of any of claims 1-5, involving a circular template.
8) The amplification process of any of claims 1-5, involving a cycling probe.
9) Any process utilizing a DNA/RNA polymerase, and a mixture nucleotides wherein at least one unnaturally base-pairing nucleotide is present.
10) The process of claim 9 wherein the mixture of nucleotides comprises both deoxyribonucleotide triphosphates and ribonucleotide triphosphates.
11) Any nucleic acid amplification or sequencing process utilizing a DNA/RNA polymerase.
12) The amplification or sequencing process of claim 11, which also utilizes a mixture of both deoxyribonucleotides and ribonucleotides wherein at least one nucleotide is an unnaturally base-pairing nucleotide.
13) An amplification template comprising one or more unnaturally base-pairing nucleotides.
14) The template of claim 13 to sequence one or more nucleotides in a target polynucleotide.
15) A primer or primer precursor comprising one or more unnaturally base-pairing nucleotides.
16) The primer or primer precursor of claim 15 used in an amplification.
17) The primer or primer precursor of claim 15 utilized to sequence one or more nucleotides in a target polynucleotide.
18) An insertion primer comprising a modification, such as a peptide nucleic acid sequence, which enhances it binding to the strand of a double stranded nucleic acid.
19) The insertion primer of 18, wherein it binds to the terminus of one of the strands of a double stranded nucleic acid.
20) The insertion primer of 18, utilized in a nucleic acid amplification.
21) A polynucleotide comprising both deoxyribonucleotides and ribonucleotides, wherein at least one nucleotide is an unnaturally base-pairing nucleotide.
Conceivably a nicking enzyme could be designed,
perhaps by mutation or shuffling known restriction or
nicking enzymes, which specifically recognizes and
nicks a sequence comprising one or more unnaturally
base-pairing nucleotides.
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Addendum 4/24/05
While the use of unnatural bases appears required in the vast majority of naturally occuring sequences, for synthetic amplification templates unnatural nucleotides are not needed. For example, 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, either while duplex (RNase H) or after displacement (single-stranded dependent RNase).
Notice also that a DNA/RNA polymerase could conceivably be replaced by a carefully composed combination of DNA polymerase and RNA polymerase.
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References
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8)
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