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 determining the presence or absence of a plurality of target nucleic acids in a composition, which comprises:
(a) preparing amplicons of the target nucleic acids by amplifying the target nucleic acids, or portions thereof, under amplification conditions;
(b) contacting the amplicons in solution with a set of oligonucleotides under hybridization conditions, wherein:
(i) each oligonucleotide in the set comprises a hybridization sequence capable of specifically hybridizing to one amplicon under the hybridization conditions when the amplicon is present in the solution,
(ii) each oligonucleotide in the set comprises a mass distinguishable tag located 5′ of the hybridization sequence,
(iii) the mass of the mass distinguishable tag of one oligonucleotide detectably differs from the masses of mass distinguishable tags of the other oligonucleotides in the set; and
(iv) each mass distinguishable tag specifically corresponds to a specific amplicon and thereby specifically corresponds to a specific target nucleic acid;
(c) generating extended oligonucleotides that comprise a capture agent by extending oligonucleotides hybridized to the amplicons by one or more nucleotides, wherein one of the one or more nucleotides is a terminating nucleotide and one or more of the nucleotides added to the oligonucleotides comprises the capture agent;
(d) contacting the extended oligonucleotides with a solid phase under conditions in which the capture agent interacts with the solid phase;
(e) releasing the mass distinguishable tags in association with the extended oligonucleotides that have interacted with the solid phase from the solid phase by competition with a competitor; and
(f) detecting the mass distinguishable tags released in (e) by mass spectrometry; whereby the presence or absence of each target nucleic acid is determined by the presence or absence of the corresponding mass distinguishable tag.
2 . The method of claim 1 , wherein competition with a competitor comprises contacting the solid phase with a competitor.
3 . The method of claim 1 , wherein the competitor consists of free capture agent, or a competing fragment or multimer thereof.
4 . The method of claim 1 , wherein the terminating nucleotide is selected from ddATP, ddGTP, ddCTP, ddTTP and ddUTP.
5 . The method of claim 1 , wherein the terminating nucleotide comprises the capture agent.
6 . The method of claim 1 , wherein the one or more nucleotides in (c) comprise one or more non-terminating nucleotides.
7 . The method of claim 6 , wherein the one or more non-terminating nucleotides comprise no capture agent.
8 . The method of claim 1 , wherein the capture agent comprises biotin or a biotin analogue, the solid phase comprises streptavidin and the competitor comprises free biotin or a biotin analogue.
9 . The method of claim 1 , wherein the releasing in (e) is carried out under elevated temperature conditions.
10 . The method of claim 9 , wherein the elevated temperature conditions comprise treatment for about 5 minutes at about 90 degrees Celsius.
11 . The method of claim 1 , wherein (c) is carried out in one container and the method further comprises transferring the released mass distinguishable tags to another container between (e) and (f).
12 . The method of claim 1 , wherein a solution containing amplicons produced in (a) is treated with an agent that removes terminal phosphates from any nucleotides not incorporated into the amplicons.
13 . The method of claim 12 , wherein the agent is a phosphatase.
14 . The method of claim 13 , wherein the phosphatase is alkaline phosphatase.
15 . The method of claim 14 , wherein the alkaline phosphatase is shrimp alkaline phosphatase.
16 . The method of claim 1 , wherein the hybridization sequence is about 5 nucleotides to about 200 nucleotides in length.
17 . The method of claim 1 , wherein the presence or absence of about 1 to about 50 or more target nucleic acids is detected.
18 . The method of claim 1 , wherein the detecting in (f) comprises a signal to noise ratio greater than the signal to noise ratio for a method in which releasing does not comprise competition with a competitor.
19 . The method of claim 1 , wherein the terminating nucleotide comprise an acyclic terminator.
20 . The method of claim 1 , wherein the capture agent comprises desthiobiotin.