Detection and quantification of biomolecules using mass spectrometry
View Patent ↗The present invention is directed in part to a method for detecting a target nucleic acid using detector oligonucleotides detectable by mass spectrometry. This method takes advantage of the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed oligonucleotide probes from hybridized duplexes and releases labels for detection by mass spectrometry. This process is easily incorporated into a polymerase chain reaction (PCR) amplification assay. The method also includes embodiments directed to quantitative analysis of target nucleic acids.
1. An amplification method of detecting the presence or absence of a target nucleic acid sequence in a sample, comprising the steps of:
(a) contacting a sample comprising a target nucleic acid sequence with a set of oligonucleotide primers comprising a 3′ end and a 5′ end, wherein a first primer comprises a sequence complementary to a region in one strand of the target nucleic acid sequence, and a second primer comprises a sequence complementary to a region in a second strand of the target nucleic acid sequence;
(b) further contacting the sample with at least one detector oligonucleotide, comprising a 3′ end and a 5′ end, and comprising a sequence complementary to a region of the target nucleic acid between the region of the target sequence complementary to the sequence of the first primer and the region of the target sequence complementary to the sequence of the second primer, which comprises a non-cleavable nucleotide incorporated at its 5′ end and a contiguous nucleotide sequence that is non-complementary to the target nucleic acid sequence linked to the 5′ end of the sequence complementary the target nucleic acid sequence, thereby forming a mixture of duplexes under hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the primer oligonucleotides and to the detector oligonucleotide such that the 3′ end of each primer oligonucleotide is upstream of the 5′ end of the detector oligonucleotide annealed to the same nucleic acid strand;
(c) exposing the products of step (b) to a template-dependent polymerizing agent having 5′ to 3′ nuclease activity under conditions permissive for amplification, whereby the primer oligonucleotides are extended to produce extension products and the detector oligonucleotide is cleaved and releases a fragment comprising the nucleotide sequence that is non-complementary to the target nucleic acid sequence, thereby producing a mass-distinguishable product; and
(d) detecting the presence or absence of the mass-distinguishable by mass spectrometry, thereby detecting the presence or absence of the target sequence in the sample.
2. The method of claim 1 , wherein steps a) and b) are performed simultaneously in a single, closed reaction vessel.
3. The method of claim 1 , wherein the detector oligonucleotide is non-extendable by the template-dependent polymerizing agent.
4. The method of claim 1 , wherein two or more target nucleic acids are detected in a single, multiplexed reaction.
5. The method of claim 1 , wherein more than one detector oligonucleotide is used to detect more than one target nucleic acid in a multiplexed reaction.
6. The method of claim 1 , wherein the target nucleic acid comprises a mixture of nucleic acid.
7. The method of claim 6 , wherein the mixture of nucleic acid comprises maternal and fetal nucleic acid, further wherein said mixture of nucleic acid has been obtained from a sample from a pregnant female.
8. The method of claim 1 , wherein the non-cleavable nucleotide is a locked nucleic acids (LNAs).
9. The method of claim 1 , wherein the mass-distinguishable product is capable of binding to a solid support upon release.
10. The method of claim 1 , wherein the mass-distinguishable products is amplified after its release.
11. The method of claim 1 , wherein the detector oligonucleotide selectively binds to a methylation-specific sequence based on the methylation status of the target nucleic acid.
12. The method of claim 1 , wherein the mass spectrometry is selected from the group consisting of MALDI-TOF MS, Tandem MS, ESI-TOF, ESI-iontrap, LC-MS, GC-MS and LOI-MS.
13. The method of claim 1 , wherein the detection is done in real-time.
14. The method of claim 1 , wherein a competitor template nucleic acid is introduced, and further wherein the competitor template nucleic acid serves as an internal control.
15. The method of claim 1 , wherein the quantity of the target nucleic acid sequence in the sample is determined.
16. The method of claim 1 , wherein the detector oligonucleotide comprises a minor groove binding moiety.
17. The method of claim 1 , wherein there are more than one non-cleavable nucleotides incorporated at the 5′ end of the complementary sequence of the detector oligonucleotide.
18. The method of claim 17 , wherein the more than one non-cleavable nucleotides are adjacent to each other.