Methods of making oligonucleotide probes
The present invention relates to methods of making linear nucleic acid probes using rolling circle amplification methods. In one aspect, an oligonucleotide template sequence is contacrted with one or more first primers, a first polymerase and first nucleotides under conditions that extend one or more hybridize primers to make a strand complementary to the oligonucleotide template sequence thereby forming a double stranded oligonucleotide. The double stranded oligonucleotide is circularized. A rolling circle template is obtained from the circularized double stranded oligonucleotide. The rolling circle template is contacted with one or more second primers, a second polymerase and second nucleotides under conditions that form a single stranded nucleic acid, and the single stranded nucleic acid is cleaved to form the plurality of nucleic acid probes.
1. A method of making a plurality of single stranded nucleic acid probes comprising:
(a) contacting a single stranded oligonucleotide template sequence that is complementary to a target sequence with a first primer comprising a polymerase recognition site and a cleavage site, a second primer comprising a label addition site, a first polymerase and first nucleotides;
(b) performing amplification to extend the first and second primers to produce a double stranded oligonucleotide comprising the single stranded oligonucleotide template sequence and its complementary sequence which is flanked by the polymerase recognition site and the cleavage site at one end, and the label addition site at the other end;
(c) circularizing the double stranded oligonucleotide to produce a circularized double stranded oligonucleotide via ds DNA ligase;
(d) obtaining a rolling circle template from the circularized double stranded oligonucleotide;
(e) contacting the rolling circle template with one or more additional, a second polymerase and second nucleotides under conditions that form a single stranded nucleic acid concatamer; and
(f) cleaving the single stranded nucleic acid concatamer to form the plurality of single stranded nucleic acid probes, wherein the cleaving is performed using the polymerase recognition site, and wherein each probe comprises a first nucleic acid sequence complementary to the target sequence and further comprises a second nucleic acid sequence complementary to the label addition site.
2. The method of claim 1 wherein the single stranded nucleic acid concatamer is DNA.
3. The method of claim 1 wherein the first polymerase or second polymerase is a DNA polymerase.
4. The method of claim 1 wherein the single stranded nucleic acid concatamer is RNA.
5. The method of claim 1 wherein the first polymerase or the second polymerase is an RNA polymerase.
6. The method of claim 1 wherein the polymerase recognition site is an RNA polymerase recognition site.
7. The method of claim 1 wherein the polymerase recognition site is a DNA polymerase recognition site.
8. The method of claim 1 wherein the single stranded nucleic acid concatamer is cleaved into a plurality of nucleic acid probes using an endonuclease.
9. The method of claim 1 wherein the single stranded nucleic acid concatamer is cleaved into a plurality of nucleic acid probes by hybridizing an oligonucleotide to the polymerase recognition site and using an endonuclease that recognizes DNA/RNA hybrids.
10. The method of claim 1 wherein the single stranded nucleic acid concatamer is cleaved into a plurality of nucleic acid probes using a mechanical shear force.
11. The method of claim 1 wherein the single stranded nucleic acid concatamer is cleaved into a plurality of nucleic acid probes using a ribozyme.
12. The method of claim 1 wherein a secondary probe comprising the label addition site is hybridized to the single stranded nucleic acid probes having the second nucleic acid sequence complementary to the label addition site.
13. The method of claim 12 wherein the secondary probe further comprises a label.