Products and processes for nucleic acid detection and quantification
Provided herein are products and processes for primer extension reactions using chain terminating reagents and enzymes having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides are not digested. Also provided herein are products and processes for the detection of the presence, absence or amount of one or more variants of a target nucleic acid species or a plurality of target nucleic acid species comprising primer extension reactions using chain terminating reagents and enzymes having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides are not digested.
1. A method for detecting the presence, absence or amount of variants of one or more target nucleic acid species or amplicons thereof, comprising:
(a) contacting a sample comprising one or more target nucleic acid species or amplicons thereof with one or more primer oligonucleotides, wherein each primer oligonucleotide comprises a region that corresponds to a portion of a target nucleic acid species or amplicon thereof and wherein each of the one or more target nucleic acid species or amplicon thereof (1) comprises at least two variants and (2) each of the one or more primer oligonucleotides is specific for a target nucleic acid species or amplicon thereof and is capable of hybridizing to the variants of the target nucleic acid species or amplicon thereof at a position 5′ to a single base position that differs between each of the variants of the target nucleic acid species or amplicon thereof, thereby generating hybridized oligonucleotides;
(b) contacting the hybridized oligonucleotides with an extension composition comprising one or more chain terminating reagents each specific for a variant of a target nucleic acid species or amplicon thereof under extension conditions, wherein the hybridized primer oligonucleotides are each extended up to, or through, the nucleotide position that is different between the variants of a corresponding target nucleic acid species or amplicon thereof, thereby generating extended primer oligonucleotides that each comprise a chain terminating reagent specific for one or more variants of the corresponding target nucleic acid species or amplicon thereof;
(c) contacting the products of (b) with an enzyme having 3′ to 5′ exonuclease activity, whereby unextended primer oligonucleotides are digested and extended primer oligonucleotides comprising a chain terminating reagent are not digested; and
(d) without subjecting the products of (b) or the products of (c) to selective capture of the extended primer oligonucleotides from the mixture of extended primer oligonucleotides and unextended primer oligonucleotides in (b) or (c), analyzing detecting the presence, absence or amount of the extended primer oligonucleotides, thereby detecting the presence, absence or amount of variants of the one or more target nucleic acid species or amplicons thereof in the sample.
2. The method of claim 1 , wherein at least one chain terminating reagent, when incorporated into a primer oligonucleotide in (b), blocks the 3′ to 5′ exonuclease activity of the enzyme.
3. The method of claim 2 , wherein at least one chain terminating reagent is a chain terminating nucleotide or all of the one or more chain terminating reagent(s) is/are a chain terminating nucleotide.
4. The method of claim 3 , wherein the chain terminating reagent(s) is/are a dideoxynucleotide or an acyclic nucleotide.
5. The method of claim 4 , wherein the dideoxynucleotide is selected from among ddATP, ddGTP, ddCTP, ddTTP and ddUTP and the acyclic nucleotide is selected from among acyATP, acyCTP, acyGTP, acyTTP and acy-Bromo-UTP.
6. The method of claim 1 , wherein the extension composition comprises two, three or four chain terminating reagents in equimolar concentrations.
7. The method of claim 1 , wherein:
(1) each of the one or more target nucleic acid species or amplicons thereof comprises a low-abundance variant and a high-abundance variant;
(2) each of the one or more primer oligonucleotides of (a) is specific for a target nucleic acid species or amplicon thereof and hybridizes to both the low-abundance variant and the high-abundance variant of the target nucleic acid species or amplicon thereof at a position 5′ to a single base position that differs between the low-abundance variant and the high-abundance variant of the target nucleic acid species, thereby generating hybridized oligonucleotides;
(3) in (b), the hybridized oligonucleotides of (2) are contacted with an extension composition comprising a chain terminating reagent specific for the low-abundance variants under extension conditions; wherein the primer oligonucleotides that hybridize to the low-abundance variant are extended by the chain terminating reagent, thereby generating extended oligonucleotides, and the primer oligonucleotides that hybridize to the high-abundance variant are not extended;
(4) in (c), the products of (3) are contacted with the enzyme having 3′ to 5′ exonuclease activity, whereby the unextended primer oligonucleotides are digested and the extended primer oligonucleotides are not digested; and
(5) in (d), the presence, absence or amount of the extended primer oligonucleotides of (4) is detected, thereby detecting the presence, absence or amount of variants of the target nucleic acid species or amplicons thereof.
8. The method of claim 1 , wherein:
(1) the sample comprises a plurality of target nucleic acid species or amplicons thereof, wherein each target nucleic acid species or amplicon thereof has a low-abundance variant and a high-abundance variant;
(2) the nucleic acids of (a) are hybridized with a plurality of primer oligonucleotides, and each primer oligonucleotide is specific for a target nucleic acid species or amplicon thereof and hybridizes to both the low-abundance variant and the high-abundance variant of the target nucleic acid species or amplicon thereof at a position 5′ to a single base position that differs between the low-abundance variant and the high-abundance variant of the target nucleic acid species, thereby generating hybridized oligonucleotides, wherein:
(i) the nucleotide at the single base position is the same or different for each of the high-abundance variants of the plurality of target nucleic acid species,
(ii) the nucleotide at the single base position is the same for each of the low-abundance variants of the plurality of target nucleic acid species, and
(iii) none of the nucleotides at the single base position for the high-abundance variants of the plurality of target nucleic acid species is the same as the nucleotide at the single base position for the low-abundance variants of the target nucleic acid species,
(3) in (b), the hybridized oligonucleotides of (2) are contacted with an extension composition comprising a chain terminating reagent specific for the low-abundance variants and one, two or three chain terminating reagents specific for one or more of the high-abundance variants under extension conditions, wherein:
(i) the one, two or three chain terminating reagents specific for one or more high-abundance variants are each at a concentration less than the concentration of the chain terminating reagent specific for the low-abundance variants, and
(ii) the extension conditions comprise multiple thermal cycles, thereby generating extended primer oligonucleotides comprising a chain terminating reagent specific for the low-abundance variants of the plurality of target nucleic acid species and/or extended primer oligonucleotides comprising a chain terminating reagent specific for the high-abundance variants of the target nucleic acid species;
(4) in (c), the products of (3) are contacted with the enzyme having 3′ to 5′ exonuclease activity, whereby the unextended primer oligonucleotides are digested and the extended primer oligonucleotides comprising a chain terminating reagent are not digested; and
(5) in (d), the presence, absence or amount of the extended primer oligonucleotides of (4) is detected, thereby detecting the presence, absence or amount of variants of the target nucleic acid species or amplicons thereof.
9. The method of claim 1 , wherein a plurality of target nucleic acid species or amplicons thereof are detected in a single, multiplexed reaction.
10. The method of claim 1 , wherein the enzyme is TTHB178.
11. The method of claim 1 , wherein the primer oligonucleotides hybridized with the target nucleic acid species or amplicons thereof do not include a detectable label attached thereto and the chain terminating reagent(s) do not include a detectable label attached thereto.
12. The method of claim 1 , wherein (d) does not include detection of a detectable label attached to the primer oligonucleotide(s) that are extended or attached to a chain terminating reagent.
13. The method of claim 1 , wherein the extended primer oligonucleotides are analyzed by mass spectrometry.
14. The method of claim 1 , wherein:
(1) the one or more target nucleic acids comprises a plurality of target nucleic acid species or amplicons thereof, wherein each species or amplicon thereof has a low-abundance variant and a high-abundance variant;
(2) the target nucleic acid species or amplicons thereof of (a) are hybridized with a plurality of primer oligonucleotides, and each primer oligonucleotide is specific for a target nucleic acid species or amplicon thereof and hybridizes to both the low-abundance variant and the high-abundance variant of the target nucleic acid species or amplicon thereof at a position 5′ to a single base position that differs between the low-abundance variant and the high-abundance variant of the target nucleic acid species, thereby generating hybridized oligonucleotides, wherein:
(i) the nucleotide at the single base position is the same for each of the high-abundance variants of the plurality of target nucleic acid species,
(ii) the nucleotide at the single base position is the same or different for each of the low-abundance variants of the plurality of target nucleic acid species, and
(iii) none of the nucleotides at the single base position for the low-abundance variants of the plurality of target nucleic acid species is the same as the nucleotide at the single base position for the high-abundance variants of the target nucleic acid species;
(3) in (b), the hybridized oligonucleotides of (2) are contacted with an extension composition comprising a chain terminating reagent specific for the high-abundance variants and one or more chain terminating reagents specific for one or more of the low-abundance variants under extension conditions, thereby generating extended primer oligonucleotides comprising a chain terminating reagent specific for the low-abundance variants of the plurality of target nucleic acid species and/or extended primer oligonucleotides comprising a chain terminating reagent specific for the high-abundance variants of the target nucleic acid species wherein:
(i) the high-abundance variants share a common chain terminating reagent that is specific for the high-abundance variants and is not specific for the low-abundance variants,
(ii) each of the low-abundance variants has a chain terminating reagent that is specific for the low-abundance variant and is not specific for the high-abundance variant, wherein the chain terminating reagent that is specific for the low-abundance variant either: (A) is unique for a particular low-abundance variant in the composition and is not shared by the other low-abundance variants in the composition, or (B) at least one of the low-abundance variants share a common chain terminating reagent with at least one other low-abundance variant in the composition, and
(iii) the concentration of the chain terminating reagent specific for the high-abundance variants is less than the concentration of the chain terminating reagent(s) specific for the low-abundance variants;
(4) in (c), the products of (3) are contacted with the enzyme having 3′ to 5′ exonuclease activity, whereby the unextended primer oligonucleotides are digested and the extended primer oligonucleotides comprising a chain terminating reagent are not digested; and
(5) in (d), the presence, absence or amount of the extended primer oligonucleotides of (4) is detected, thereby detecting the presence, absence or amount of variants of the target nucleic acid species or amplicons thereof.
15. The method of claim 1 , wherein the target nucleic acid species or amplicons thereof, the one or more primer oligonucleotides, the products of (b) and the products of (c) are not captured by binding to a solid phase.
16. The method of claim 1 , wherein the target nucleic acid species or amplicons thereof, primer oligonucleotides, extended primer oligonucleotides and chain terminating reagents do not comprise a capture agent or member of a binding pair for which the other member of the binding pair is linked to a solid phase.
17. The method of claim 1 , wherein the target nucleic acid species or amplicons thereof of the sample are hybridized in (a) to a plurality of primer oligonucleotides each comprising a region that corresponds to a portion of one of a plurality of target nucleic acids.
18. The method of claim 7 , wherein the target nucleic acid species or amplicons thereof, the one or more primer oligonucleotides, the products of (3) and the products of (4) are not captured by binding to a solid phase.
19. The method of claim 8 , wherein the target nucleic acid species or amplicons thereof, the plurality of primer oligonucleotides, the products of (3) and the products of (4) are not captured by binding to a solid phase.
20. The method of claim 14 , wherein the plurality of primer oligonucleotides, the products of (3) and the products of (4) are not captured by binding to a solid phase.