Products and processes for multiplex nucleic acid identification
Provided herein are products and processes for detecting the presence or absence of multiple target nucleic acids. Certain methods include amplifying the target nucleic acids, or portion thereof; extending oligonucleotides that specifically hybridize to the amplicons, where the extended oligonucleotides include a capture agent; capturing the extended oligonucleotides to a solid phase via the capture agent; releasing the extended oligonucleotide by competition with a competitor; detecting the extended oligonucleotide, and thereby determining the presence or absence of each target nucleic acid by the presence or absence of the extended oligonucleotide.
1. A method for detecting the presence, absence or amount of genetic variants in a composition, comprising:
(a) obtaining a plurality of target nucleic acids and/or amplicons thereof, or portions thereof, wherein there are two or more possible variants for each target nucleic acid and at least a first variant of the two or more possible variants, if present, is of lower abundance than a second variant of the two or more possible variants;
(b) hybridizing the target nucleic acids and/or amplicons thereof to oligonucleotide species, wherein each oligonucleotide species is capable of hybridizing to a target nucleic acid, thereby generating hybridized oligonucleotide species;
(c) contacting the hybridized oligonucleotide species with an extension composition consisting of two terminating nucleotides specific for one or more of the first variants or consisting of three terminating nucleotides specific for one or more of the first variants and no terminating nucleotides specific for the second variants under extension conditions; wherein:
(i) the terminating nucleotides each comprise a capture agent, and
(ii) the oligonucleotide species hybridized to the first variant are extended by a terminating nucleotide and the oligonucleotide species hybridized to the second variant are not extended by a terminating nucleotide, thereby generating extended oligonucleotide species;
(d) capturing the extended oligonucleotide species to a solid phase that captures the capture agent;
(e) releasing the captured extended oligonucleotide species from the solid phase; and
(f) detecting the mass of each extended oligonucleotide species released from the solid phase in (e) by mass spectrometry, whereby the presence, absence or amount of the low-abundance genetic variants is detected.
2. The method of claim 1 , wherein releasing in (e) comprises contacting the solid phase in (d) with a releasing agent that has a higher affinity for the solid phase than the capture agent.
3. The method of claim 1 , wherein the capture agent comprises biotin or a biotin analogue.
4. The method of claim 3 , wherein the solid phase comprises avidin or a modified form of avidin or streptavidin or a modified form of streptavidin.
5. The method of claim 4 , wherein the releasing agent comprises free biotin or a biotin analogue.
6. The method of claim 4 , wherein the modified form of avidin is nitroavidin or derivatives thereof.
7. The method of claim 4 , wherein the modified form of streptavidin is nitrostreptavidin or derivatives thereof.
8. The method of claim 3 , wherein releasing in (e) comprises contacting the solid phase in (d) with a releasing agent comprising free biotin or a biotin analogue at a concentration from about 10 to about 100 ug/ml.
9. The method of claim 3 , wherein releasing in (e) comprises contacting the solid phase in (d) with a releasing agent comprising free biotin or a biotin analogue under elevated temperature conditions of about 90° C. to about 95° C. for about 1 to about 5 minutes.
10. The method of claim 3 , wherein releasing in (e) comprises contacting the solid phase in (d) with a releasing agent comprising free biotin or a biotin analogue under elevated temperature conditions of about 95° C. for about 5 minutes or about 90° C. for about 5 minutes.
11. The method of claim 3 , wherein the biotin analogue is selected from among 9-methylbiotin, biotin methyl ester (MEBio), desthiobiotin (DEBio), 2′-iminobiotin (IMBio), e-N-Biotinyl-L-lysine, diaminobiotin (DABio), biotin sulfone, 2′-thiobiotin and N3′-ethyl biotin.
12. The method of claim 1 , wherein the terminating nucleotides independently are selected from ddATP, ddGTP, ddCTP, ddTTP and ddUTP.
13. The method of claim 1 , wherein the lower abundance variant, if present, is about 10% or less of the target nucleic acid.
14. The method of claim 1 , wherein the lower abundance variant, if present, is about 5% or less of the target nucleic acid.
15. The method of claim 1 , wherein the plurality of target nucleic acids is about 2 to about 20 target nucleic acids.
16. The method of claim 1 , wherein a signal to noise ratio and/or a sensitivity for extending only a first variant is greater than a signal to noise ratio and/or a sensitivity for extending a first variant and a second variant.
17. The method of claim 1 , wherein the lower abundance variant, if present, is about 1% or less of the target nucleic acid.