Methods of forming circular nucleic acid probes and uses thereof
The present invention relates generally to a method of amplifying closed circular nucleic acid probes and, more particularly, to a method of amplifying closed circular nucleic acid probes by rolling circle amplification. The method of the present invention is useful in a range of applications involving the detection of nucleic acid sequences such as, but not limited to, the identification of genetic disorders, genetic variants or the presence of microbiological or viral agents.
1. A method for forming a circular nucleic acid probe comprising
contacting a first target nucleic acid sequence molecule and a second target nucleic acid sequence molecule with a first nucleic acid probe and a second nucleic acid probe, wherein the nucleic acid probes each comprise a 5′ target probe region and a 3′ target probe region, wherein the 5′ target probe region of the first nucleic acid probe hybridizes to the first target nucleic acid sequence molecule, wherein the 3′ target probe region of the first nucleic acid probe hybridizes to the second target nucleic acid sequence molecule, wherein the 3′ target probe region of the second nucleic acid probe hybridizes to the first target nucleic acid sequence molecule, wherein the 5′ target probe region of the second nucleic acid probe hybridizes to the second target nucleic acid sequence molecule, wherein the target nucleic acid sequences of the first and second target nucleic acid sequence molecules are different,
circularizing the first and second nucleic acid probes thereby forming a circular nucleic acid probe, wherein the gap between the 5′ end of the first nucleic acid probe and the 3′ end of the second nucleic acid probe is closed and wherein the gap between the 3′ end of the first nucleic acid probe and the 5′ end of the second nucleic acid probe is closed.
2. The method of claim 1 further comprising, subjecting the circular nucleic acid probe to rolling circle amplification, wherein the rolling circle amplification uses one or more complementary primers, wherein the complementary primers are complementary to the nucleic acid probe, wherein the rolling circle amplification uses one or more sense primers, wherein the rolling circle amplification produces a nucleic acid strand complementary to the nucleic acid probe, wherein the sense primers are complementary to the nucleic acid strand complementary to the nucleic acid probe, wherein the sense primers interact with regions of the nucleic acid strand complementary to the nucleic acid probe.
3. The method of claim 2 conducted in solid phase.
4. The method of claim 1 further comprising subjecting the circular nucleic acid probe to amplification.
5. The method of claim 4 wherein said amplification is rolling circle amplification.
6. The method of claim 5 wherein said rolling circle amplification is multiple primer rolling circle amplification.