Methods and compositions for multi-stage primer extension reactions

Abstract

Methods and compositions for multi-stage primer extension reactions, such as multiplex polymerase chain reaction (PCR) and reverse transcriptase PCR are described. Primer extension stages are performed in a closed vessel without opening the closed vessel between stages. The multi-stage primer extension methods and compositions utilize early stage primers in early stages and late stage primers in later stages, where the late stage primers are blocked from extending during the early stages. The blocking primers of the present technology comprise a photo-cleavable blocking group and are substantially inactive until the blocking group is cleaved by exposure to ultraviolet light. The blocking primers can be activated by ultraviolet light without opening the vessel.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 994,989, filed March 26, 2020, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to methods and compositions for multi-stage primer extension reactions, such as multiplex polymerase chain reaction (PCR) and reverse transcriptase-PCR. Background Technology

[0004] Polymerase chain reaction (PCR) is a method for the specific amplification of DNA sequences. PCR is a useful and widely used method for the amplification of DNA targets in the preparation of Next Generation Sequencing (NGS) libraries. Specifically, primers hybridize and extend their target sequences in a mixture of nucleic acids, followed by additional rounds of primer hybridization and extension. PCR can exponentially amplify sequences between primers, making it a highly sensitive technique. However, PCR can lead to undesirable amplification products. First, off-target amplification can reduce the yield of the target sequence if primers bind to sequences other than their target. Second, the concentration of PCR primers must be much higher than the target sequence (to support exponential reactions in subsequent rounds), therefore, primers can sometimes interact with other primers, resulting in primer dimers. Third, different target sequences may amplify at different efficiencies, depending on their length, GC content, primer sequence, etc.

[0005] Multiplex PCR (mPCR) is a method that uses many (potentially hundreds or thousands) primers in the extension reaction. This is convenient because many target sequences can be amplified in the same tube, and potentially, many target sequences can be amplified from the same aliquot of the sample. However, in mPCR, off-target amplification, primer dimer formation, and uneven amplification are problems that are intertwined. In fact, the primer dimer problem can worsen exponentially because each new pair of primers added to the multiplex reaction may potentially interact with all the other primers in the mixture.

[0006] Multiplex PCR has the potential to generate significant savings in laboratory time and effort. This technique has been applied in many areas of human DNA testing, including gene deletion analysis, mutation and polymorphism analysis, quantitative analysis, and reverse transcription (RT)-PCR. In the field of infectious diseases, multiplex PCR has been used for the identification of viruses, bacteria, and parasites. However, the use of mPCR presents several challenges, including poor sensitivity, poor specificity, preferential amplification of certain specific targets, and / or amplification of unexpected sequences.

[0007] Typically, multiplex PCR occurs in two separate stages (i.e., multistage PCR). In the first stage reaction, target-specific primers with a universal sequence are used to amplify specific target polynucleotides, and universal forward and reverse sequences are added to each amplicon. The products are then purified before subsequent PCR amplification reactions to remove unreacted target-specific primers and other reagents. In subsequent PCR reactions, amplicons from the earlier stage are then amplified using universal primers designed to hybridize with the universal sequence, and any other sequences (e.g., adaptors) required for further processing and identification purposes are added.

[0008] Such systems are labor-intensive. Furthermore, competition between target-specific primers and universal primers can lead to biased amplification, necessitating stopping and purifying the reaction mixture after the early stages. This can introduce potential errors and contamination into the system. Therefore, it is desirable to determine a method that allows two stages to be performed in a single reaction vessel. Moreover, it would be particularly advantageous (e.g., for preventing contamination) and convenient if two or more stages could be performed without opening the reaction vessel.

[0009] In reverse transcriptase-polymerase chain reaction (RT-PCR), RNA is reverse transcribed into cDNA in an initial stage, and then the cDNA is amplified in a subsequent PCR step, typically using target-specific primers. RT-PCR faces the same challenges as multi-step PCR because gene-specific primers can be non-specifically initiated during cDNA synthesis, which is performed at relatively low temperatures (37–60°C). Specificity is typically improved by performing reverse transcription and PCR in two separate containers, preventing PCR primers from interacting with each other or with the RT primers used to initiate cDNA synthesis (e.g., oligomers (dT), random hexamers, or gene-specific reverse primers). However, opening the test tube between the RT and PCR steps increases labor and the risk of contamination. Summary of the Invention

[0010] This technology relates to a novel method for performing a multi-stage polymerase chain reaction in a closed container, wherein the mixture comprises: i) a polynucleotide target; ii) an early-stage primer capable of primer extension; iii) a late-stage primer containing an optically cleavable blocking group at its 3' end; iv) a primer extension enzyme; and v) other reagents optionally desired. Examples of early-stage primers include target-specific primers and reverse transcriptase (RT) primers. Target-specific primers may comprise a 5' region and a 3' region, wherein the 3' region contains a target-specific sequence and the 5' region contains a universal sequence. When such target-specific primers are used for early-stage primer extension reactions, the late-stage primers may be universal primers containing a universal sequence or a portion thereof, and an optically cleavable blocking group at its 3' end. In some embodiments, the container is sealed after preparation of the mixture, and an early polymerase chain reaction is performed with the mixture to generate a target amplicon. The universal primer is unblocked in the mixture to generate unblocked universal primers containing a universal sequence or a portion thereof. In some embodiments, the unblocking step is performed without opening the container. A later-stage primer extension reaction is performed using the unblocked primers and the target amplicon, wherein the unblocked primers amplify the target amplicon. In some embodiments, the unblocking step is performed by exposing universal primers in a sealed container to ultraviolet light.

[0011] In another aspect, this technology relates to novel compositions for performing multi-stage PCR, wherein the compositions comprise a) a polynucleotide target; b) an early-stage primer capable of primer extension; and c) a late-stage primer, the late-stage primer containing a light-cleavable blocking group at its 3' end. Examples of early-stage primers include target-specific primers and reverse transcriptase (RT) primers. Target-specific primers may comprise a 5' region and a 3' region, wherein the 3' region contains a target-specific sequence and the 5' region contains a universal sequence. When such target-specific primers are early-stage primers, the late-stage primers may be universal primers containing a universal sequence or a portion thereof, and a light-cleavable blocking group at its 3' end. In some embodiments, the compositions are contained in a container that is sealed after the composition is prepared. By exposure to ultraviolet light, the late-stage primers can be unblocked and activated for PCR amplification.

[0012] In another aspect, this technology relates to a novel method for performing multi-stage RT-PCR in a sealed container, wherein the mixture comprises: i) a polyribonucleotide (RNA) target; ii) early-stage primers comprising oligomeric (dT) primers, random primers, or target-specific RT primers for cDNA synthesis; iii) late-stage primers containing a target-specific sequence at their 5' end and a light-cleavable blocking group at their 3' end; iv) reverse transcriptase; v) DNA polymerase; and vi) other reagents optionally desired. In some embodiments, the container is sealed after preparation of the mixture, and reverse transcription is performed at a constant temperature (37-60°C) prior to PCR thermal cycling. The target-specific PCR primers are unsealed without opening the container, and a PCR step is performed with the unsealed PCR primers and cDNA, wherein the unsealed primers amplify one or more target amplicones. In some embodiments, the unsealing step is performed by exposing the sealed target-specific PCR primers in the sealed container to ultraviolet light.

[0013] In another aspect, this technology relates to novel compositions for performing multi-stage RT-PCR in a sealed container. The composition comprises a) a polynucleotide (RNA) target; b) an early-stage primer comprising an unblocking RT primer for cDNA synthesis; and c) a late-stage primer comprising a blocking primer for late-stage primer extension, wherein the late-stage primer contains a photocleavable blocking group at its 3' end. The blocked late-stage primer may be a target-specific PCR primer, a random primer, or a universal primer. The late-stage primer can be unblocked after cDNA synthesis and activated for PCR amplification by exposure to ultraviolet light. In some embodiments, the composition is contained in a container that is sealed after the composition is prepared. Attached Figure Description

[0014] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the teachings of this invention in any way.

[0015] Figure 1 A schematic diagram illustrating embodiments of the multi-stage PCR primers, methods, and compositions of the present invention is provided.

[0016] Figure 2 Reversed-phase HPLC traces were provided, demonstrating the generation of universal blocking primers with optically cleavable blocking groups at their 3' ends.

[0017] Figure 3 A schematic diagram illustrating embodiments of the multi-stage RT-PCR primers, methods, and compositions of the present invention is provided.

[0018] Figure 4 Reverse-phase HPLC traces are provided, demonstrating that the blocking universal primers of this invention in a PCR tube are unblocked by exposing the primers to 365 nm UV light for 10 seconds.

[0019] Figure 5 Images of the Bioanalyzer 2100 are provided, demonstrating that the light-cleavable blocking primers extend in PCR only after the primers are exposed to UV light.

[0020] Figure 6A and Figure 6B BioAnalyzer images of a single-vessel RT-PCR reaction are provided, wherein the single-vessel RT-PCR reaction was performed in an early stage with unblocked RT primers in the presence of blocked late-stage primers, wherein the blocked late-stage primers are unblocked in the late stage. Detailed Implementation

[0021] This technique involves multi-stage primer extension reactions, such as multiplex PCR and RT-PCR using unblocked early-stage primers and unblocked late-stage primers. The blocking primers contain a light-cleavable blocking group at their 3' end. Polynucleotide targets undergo primer extension in the early stage to form products, such as target amplicons or target cDNA. For example, genomic DNA can be amplified in the early stage using target-specific primers containing a target-specific sequence in the 3' region and a universal sequence in the 5' region. The product of this early stage contains a target amplicon containing a universal sequence at both the 5' and 3' ends. The target amplicon is then amplified in the late-stage primer extension reaction using universal primers that have been unblocked by light cleavage of the blocking group, for example, by exposure to ultraviolet light. As another example, RNA targets can undergo primer extension in the early stage to produce target cDNA. The target cDNA can then be extended in the late stage using either unblocked target-specific primers or universal primers.

[0022] Figure 1 A schematic diagram of the multi-stage PCR method of this technology is presented. The presented PCR method can be performed in a sealed container containing a polynucleotide target, a target-specific primer containing a universal sequence, and a 3'-blocked photocleavable universal primer, wherein the 3'-blocked photocleavable universal primer contains a photocleavable blocking group at its 3' end.

[0023] Figure 1This illustrates the amplification of a polynucleotide target in early-stage PCR using target-specific forward and reverse primers. The forward primer contains a target-specific sequence that will hybridize with the target, as well as a universal sequence indicated as tag 1, and the reverse primer also contains a target-specific sequence that will hybridize with the target, as well as a different universal sequence indicated as tag 2. Amplification of the polynucleotide target in early-stage PCR produces a target amplicon containing the polynucleotide target sequence and also containing universal sequences tag 1 and 2 (or their complementary sequences) at the 5' and 3' ends of the target amplicon, respectively. Early-stage PCR amplification is typically multiplex PCR amplification, in which multiple targets are amplified in parallel using multiple sets of target-specific primers. Exemplary target-specific primer sets are available from Agilent's SureMASTR technology, such as BRCA MASTR DXAssay.

[0024] Figure 1 The late-stage amplification of the target amplicon using a set of 3'-closed, optically cleavable universal primers is also demonstrated. In this technique, Figure 1 The forward universal primer shown contains a lightly cleavable blocking group at its 3' end; a 3' region containing a sequence of universal sequence tag 1 or a portion thereof; and an adapter sequence in the 5' region, the adapter sequence being indicated as adapter 1. Figure 1 The reverse universal primer shown contains a light-cleavable blocking group at its 3' end; a 3' region containing a sequence of universal sequence tag 2 or a portion thereof; a molecular identifier sequence indicated as MID1; and an adapter sequence in the 5' region indicated as adapter 2. The light-cleavable blocking groups in both the forward and reverse universal primers are indicated by a termination symbol. The blocked universal primers are present in the reaction mixture during early-stage PCR but are substantially inactive for PCR amplification until they are unblocked. The blocked universal primers can be unblocked and activated by exposure to ultraviolet light for later-stage PCR amplification. After exposure to ultraviolet light, the unblocked universal primers are active for PCR amplification. Figure 1 As shown, late-stage amplification leads to the general amplification of early-stage amplicones to produce late-stage amplicones. Therefore, the amplicon of the general amplification of the late stage contains the sequences of target-specific primers and general primers (e.g., the sequences of adaptor 1 and adaptor 2).

[0025] Because the blocked universal primers of this technology can be unblocked by exposure to ultraviolet light to allow for the addition of the universal primers after the early stages of PCR, the universal primers can be present in the mPCR reaction mixture during the early-stage PCR without interfering with the initial target amplification. Surprisingly, the blocked universal primers are essentially inactive in the early stages but active for PCR in the later stages. Furthermore, the photocleavable nature of the blocking group allows the universal primers to be unblocked and activated by exposing the entire container holding the PCR mixture to ultraviolet light. This unblocking capability is convenient and highly beneficial because it reduces or avoids the introduction of contamination, as the later-stage PCR can be performed after the early-stage PCR without opening the PCR mixture container to add the universal primers.

[0026] For example, after the early stages of PCR are completed, 3'-blocked, light-cleavable universal primers can be unblocked by exposure to ultraviolet light, such as 365 nm. Exposure to ultraviolet light can be performed in any suitable manner. Unblocking can be performed, for example, by removing the PCR container from the thermal cycler and placing it on a UV light source (e.g., a UV light chamber). Alternatively, the PCR thermal cycler can be modified to directly apply ultraviolet (UV) light. In one case, when universal primers are retained in the same sealed PCR reaction container, ultraviolet light can be applied to unblock the universal primers. The same sealed PCR reaction container can then be used for the later stages of amplification with the now-unblocked universal primers.

[0027] In another aspect, this technique relates to multi-stage RT-PCR, wherein the late-stage primers contain target-specific primers having a photocleavable blocking group at their 3' end. The polynucleotide target is reverse transcribed in the first stage by reverse transcriptase and unblocked RT primers (e.g., oligo(dT) primers, random primers, or target-specific reverse primers). The product of the early-stage primer extension reaction is target cDNA. During late-stage PCR, the target cDNA can be amplified using late-stage primers (e.g., target-specific primers that have been unblocked by photocleavage of the blocking group).

[0028] Figure 3 A schematic diagram of the multi-stage RT-PCR of this technique is shown. The presented RT-PCR can be performed in a sealed container containing a polynucleotide target, unblocked RT primers, and target-specific primers containing a 3' photocleavable blocking group. The photocleavable blocking group in the target-specific primers is indicated by a stop symbol. Figure 3 This demonstrates early-stage reverse transcription of polynucleotide targets using an unblocked reverse primer in the presence of a closed target-specific primer. Figure 3The amplicon was also shown to be amplified in the later stages after the 3' blocking group had been removed by exposure to ultraviolet light.

[0029] In some embodiments, the photocleavable blocking group of this technology is attached to a suitable reporter molecule, such as a fluorophore. In other embodiments, the photocleavable blocking group of this technology is not attached to a reporter molecule. It is contemplated that the photocleavable blocking groups of this technology will function appropriately regardless of whether they include a fluorophore; only the presence of a photocleavable blocking group is required.

[0030] Furthermore, since the photocleavable blocking group of this technology is removed by exposure to ultraviolet light, the blocking group of this invention does not affect the function of the primer extension enzyme. Therefore, the photocleavable blocking group of this technology is compatible with standard PCR and RT-PCR components (e.g., polymerase, nucleotides (dNTPs), and buffers).

[0031] Before further describing the exemplary embodiments in detail, the following definitions and explanations are set forth to illustrate and define the meaning and scope of the terms used in this specification.

[0032] Numerical ranges include the values ​​that define the range. Unless otherwise specified, nucleic acids are written from left to right with a 5' to 3' orientation; amino acid sequences are written from left to right with the orientation from amino to carboxyl groups.

[0033] Unless otherwise specified, this technique may employ techniques and descriptions within the scope of the art, including those of organic chemistry, polymer technology, molecular biology (including recombinant technologies), cell biology, biochemistry, and immunology. Such techniques include polymer array synthesis, hybridization, ligation, and the detection of hybridization using tags.

[0034] As used herein, unless the context clearly indicates otherwise, the singular forms of “a,” “a,” and “the” include plural referents. For example, the term “a primer” refers to one or more primers, i.e., a single primer and multiple primers. “Multiple” contains at least two members. In some cases, multiple can have at least 10, at least 100, at least 100, at least 10,000, at least 100,000, at least 10,000, or at least 10 6 At least 10 7 At least 10 8 One or at least 10 9 One or more members.

[0035] It should also be noted that the claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a prior basis for the use of exclusive terms such as “alone” or “only” or the use of “negative” limitations in relation to the description of the elements of the claims.

[0036] As used in the specification and appended claims, and in addition to their ordinary meaning, the terms "substantially" or "truly" mean to the extent or degree acceptable to a person skilled in the art. For example, "substantially inactive" means that a person skilled in the art would consider the level of activity to be negligible.

[0037] As used herein, the term "sample" refers to a material or mixture of materials containing one or more polynucleotides or fragments of interest. In some embodiments, the term refers to any plant, animal, or viral material containing DNA, RNA, or other polynucleotides, such as tissue or fluid isolated from patients (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections), preserved tissues (e.g., FFPE sections), or in vitro cell culture components, as well as samples from the environment. Any sample containing nucleic acids (e.g., genomic DNA from tissue culture cells or tissue samples) may be used in this technique.

[0038] As used herein, the term "nucleic acid sample" refers to a sample containing nucleic acids. Nucleic acid samples can be complex because they contain multiple different molecules with distinct sequences. Nucleic acid samples from mammals (e.g., mice or humans) are a type of complex sample. Complex samples may have more than 10 4 10 5 10 6 Or 10 7 There are several different nucleic acid molecules. Furthermore, complex samples may contain only a few molecules, wherein the total number of these molecules is greater than 10. 4 10 5 10 6 Or 10 7 One or more nucleotides. The term "complexity" generally refers to the total number of distinct sequences in a population, such as a fragment, adaptor, or a population of fragments linked by an adaptor. For example, if a population has 4 distinct sequences, the complexity of the population is 4. Depending on the desired outcome, a population can have a complexity of at least 4, at least 8, at least 16, at least 100, at least 1,000, at least 10,000, or at least 100,000 or higher.

[0039] The term "nucleotide" refers to naturally occurring nucleotides, including guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively), as well as modified pyrimidine and purine derivatives and other non-naturally occurring moieties. These modified pyrimidine and purine derivatives and other non-naturally occurring moieties contain not only known purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose, or other heterocycles. Furthermore, the term "nucleotide" includes those moieties containing haptens or fluorescent labels, and may contain not only conventional ribose and deoxyribose but also other sugars. Modified nucleotides also include modifications to the sugar moiety, for example, where one or more hydroxyl groups are replaced by halogen atoms or aliphatic groups, or functionalized into ethers, amines, etc.

[0040] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe nucleotide-containing polymers of any length (e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases) composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), and which can be produced naturally, chemically, enzymatically, or synthetically. The term includes polymers having PNA, LNA, or UNA. DNA and RNA have deoxyribose and ribose backbones, respectively, while the backbone of PNA consists of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In PNA, various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. Locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose portion of an LNA nucleotide is modified with an additional bridge connecting the 2' oxygen and 4' carbon. This bridge "locks" the ribose in a 3'-endo-(north) conformation, typically found in A-type duplexes. LNA nucleotides can be mixed with DNA or RNA residues in oligonucleotides whenever desired. The term "unstructured nucleic acid" or "UNA" refers to a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, unstructured nucleic acids can contain G' and C' residues, where these residues correspond to non-naturally occurring forms of G and C, i.e., analogs, which pair with each other with reduced stability but retain the ability to pair with naturally occurring C and G residues, respectively.

[0041] The term "base" refers to a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, typically found in nucleic acids and their natural, substituted, modified, or engineered variants or analogs, capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with appropriately complementary bases.

[0042] The term "connector" refers to one or more divalent groups that act as a molecular bridge for covalent bonding between two other groups, such as -C(O)NH-, -C(O)O-, -NH-, -S-, -S(O)n, where n is 0, 1, or 2, -O-, -OP(O)(OH)O-, -OP(O)(O - O-, alkyldiyl, alkenyldiyl, ynyldiyl, aromatic alkenyl, heteroaromatic diyl, and combinations thereof. The connector may have a side chain or a side functional group (or both).

[0043] The term "reporter molecule" refers to a chemical component that can generate a detectable signal, directly or indirectly. Examples of reporter molecules include fluorescent dye groups, radioactive labels, or groups that affect the signal through chemiluminescence or bioluminescence. Examples of fluorescent dye groups include guar tannin, fluorescein, rhodamine, botrypol, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, ALEXA FLUOR 350, ALEXA FLUOR 405, ALEXA FLUOR 430, ALEXA FLUOR 488, ALEXA FLUOR 514, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 555, ALEXA FLUOR 568, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 610, ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 680, ALEXA FLUOR 700, ALEXA FLUOR 750, and squaric acid dyes. Other examples of fluorescent dye reporter molecules that may be used in some embodiments of the present invention are disclosed in Haugland, 2005 and U.S. Patents 4,439,356 and 5,188,934, which are incorporated herein by reference. Examples of radiolabels that may be used as reporter molecules in some embodiments of the present invention are well known in the art, for example… 35 S, 3 H, 32 P or 33P. Examples of reporter molecules that function by means of chemiluminescence or bioluminescence and can be used as reporter molecules in some embodiments of the present invention are described in Nieman, 1989; Given & Schowen, 1989; Orosz et al., 1996; and Hastings, 1983, which are incorporated herein by reference.

[0044] As used herein, the term "oligonucleotide" refers to a single-stranded polymer of nucleotides, typically ranging from about 2 to 200 nucleotides in length, and typically up to 500 nucleotides. Oligonucleotides can be synthetic or enzymatically prepared, and in some embodiments are 30 to 150 nucleotides in length. Oligonucleotides can contain ribonucleotide monomers (i.e., oligoribonucleotides) or deoxyribonucleotide monomers, or both. In some embodiments, the oligonucleotides of the present invention can be, for example, 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, or 150 to 200 nucleotides in length.

[0045] The term "primer" refers to a natural or synthetic oligonucleotide that can act as an initiation site for nucleic acid synthesis when forming a double helix with a polynucleotide template (e.g., a polynucleotide target) and extends from its 3' end along the template to form an extended double helix. The term "extension," as used herein, refers to the extension of a primer by adding nucleotides using a primer extension enzyme. If a primer annealed with a nucleic acid is extended, the nucleic acid acts as a template for the extension reaction. The sequence of the nucleotides added during the extension process is determined by the sequence of the polynucleotide template. Primers can be extended using primer extension enzymes such as DNA polymerase and reverse transcriptase. Reverse transcriptase is an RNA-dependent DNA polymerase that incorporates a deoxynucleotide opposite to the RNA template. The resulting cDNA (complementary DNA) can then serve as a DNA template in late-stage PCR using a DNA-dependent DNA polymerase. Primer length is typically compatible with its intended use in the synthesis of primer extension products and is generally between 8 and 100 nucleotides, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, etc., more typically between 18-40, 20-35, 21-30 nucleotides, and any length within these ranges. Typical primer lengths can be between 10 and 50 nucleotides, such as 15-45, 18-40, 20-30, 21-25, etc., and any length within these ranges. In some implementations, the primer length is typically no more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides.

[0046] Primers used for amplification are typically single-stranded, but can also be provided to the mixture in double-stranded form. If double-stranded, the primers are usually first treated to separate their strands before being used to prepare the extension product. Thus, the primers are complementary to the template and form a complex via hydrogen bonding or hybridization with the template to obtain a primer / template complex for initiation of synthesis via polymerase, which is extended during DNA synthesis by adding a covalently bonded base complementary to the template and attached to its 3' end. The terms "reverse primer" and "forward primer" refer to primers that hybridize to different strands in a double-stranded DNA molecule, where the extension of the primer by polymerase is in the direction toward the other primer. cDNA synthesis can be initiated by reverse transcriptase (RT) primers. For example, oligonucleotides containing a series of deoxythymidine nucleotides (oligomeric (dT)) can anneal to the 3' poly-A tail of the RNA transcript. Alternatively, RT primers can anneal to multiple sequence-specific sites within the RNA (target-specific primers). Random primers can also be used as RT primers.

[0047] A "pair" of primers refers to forward and reverse primers designed to amplify a double-stranded polynucleotide target. In some embodiments, the compositions, methods, and kits of the present invention comprise highly multiplexed target-specific primer sets, such as at least 5 pairs, or at least 10 pairs, or at least 20 pairs, or at least 50 pairs, or at least 100 pairs, or at least 200 pairs, or at least 500 pairs, or at least 1,000 pairs, or at least 2,000 pairs, or at least 5,000 pairs, or at least 10,000 pairs, or at least 20,000 or more pairs of target-specific primers.

[0048] The term "primer extension reagent" refers to any reagent required or suitable for use in performing primer extension reactions (e.g., polymerase chain reaction (PCR)) on polynucleotide molecules, such as polynucleotide targets. Primer extension reagents typically consist of primers, a heat-stable polymerase or reverse transcriptase, and a mixture of nucleotides and appropriate buffers. Depending on the enzyme used, ions (e.g., Mg2+) may also be present. 2+ cDNA synthesis is initiated by a reverse primer, which anneals to the 3' poly-A tail of the RNA transcript (oligomeric (dT)) or to multiple sequence-specific sites within the RNA (randomizer, target-specific primer).

[0049] As used herein, the term "universal sequence" refers to a sequence common to two or more nucleic acid molecules in a set or population, preferably a sequence common to substantially all nucleic acid molecules in a set or population, wherein the nucleic acid molecules also have portions that are different from each other (e.g., the target portion in a set of polynucleotide target amplicon). Universal sequences can be present in different members of a set or population of molecules, thereby allowing for the co-processing of different molecules. Non-limiting examples of universal sequences include sequences that are identical to or complementary to the capture sequence of a flow cell. Similarly, universal sequences can allow for the amplification of multiple different nucleic acids using a universal amplification primer set complementary to a portion of the universal sequence (e.g., a universal primer binding site).

[0050] In some embodiments, the target-specific primers currently described have a 5' region containing a universal sequence, such that the universal sequence or its complementary sequence can be incorporated into the target amplicon generated in an early PCR stage. Subsequent amplification in a later PCR stage using a deblocked universal primer that has hybridized with the universal sequence can be used to universally amplify the target amplicon present in the PCR mixture.

[0051] The terms "upstream" and "...of 5'" referring to positions within a nucleic acid sequence are used interchangeably to refer to the relative position closer to the 5' end of the sequence. Similarly, the terms "downstream" and "...of 3'" referring to positions within a nucleic acid sequence are used interchangeably to refer to the relative position closer to the 3' end of the sequence.

[0052] As used herein, the term "hybridization" refers to any process in which a nucleic acid strand binds to a complementary strand through base pairing. The term "hybridization" also encompasses processes in which a nucleic acid strand anneals to a second complementary nucleic acid strand under normal hybridization conditions to form a stable duplex (homoduplex or heteroduplex), and does not form a stable duplex with unrelated nucleic acid molecules under the same normal hybridization conditions. The terms "duplex" or "double-stranded" as used herein describe two complementary polynucleotides that have paired bases (i.e., hybridized together). Duplex formation is accomplished by annealing two complementary nucleic acid strands in a hybridization reaction. The hybridization process can become highly specific by modulating the hybridization conditions (often referred to as hybridization strictness) so that hybridization between the two nucleic acid strands will not form a stable duplex unless the two nucleic acid strands contain a certain number of substantially or completely complementary nucleotides in a specific sequence. For any given hybridization reaction, "normal hybridization" or "normal strict conditions" are readily determined.

[0053] The term "complementary" refers to two nucleic acids that hybridize with each other under highly stringent conditions. The term "perfectly complementary" refers to a double helix in which each base of one nucleic acid pairs with a complementary nucleotide base in the other nucleic acid. In many cases, complementary sequences have at least 10, for example, at least 12 or 15 complementary nucleotides. Conversely, if two nucleic acids are "non-complementary," they will not hybridize with each other, but some sequence matching, i.e., less than 100% non-complementarity, is permissible, as long as the two strands remain in single-stranded form when used in the method of the invention and under the conditions defined above.

[0054] The term "amplification" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid (e.g., a polynucleotide target). Amplifying a nucleic acid molecule may include denaturing the template nucleic acid, annealing the primers to the template nucleic acid at a temperature below the primer melting temperature, and enzymatically extending the primers to produce an amplification product. The denaturation, annealing, and extension steps may be performed once or multiple times. In some cases, the denaturation, annealing, and extension steps are performed multiple times, resulting in a continuous increase in the amount of amplification product, often exponentially, although the method of the present invention does not require exponential amplification. Amplification typically requires the presence of deoxyribonucleoside triphosphates, DNA polymerase, and appropriate buffers and / or cofactors for achieving optimal polymerase activity. The terms "amplification product" or "amplifier" refer to the nucleic acid sequence produced by an amplification process as defined herein. Reverse transcription is a linear amplification reaction that uses a specialized DNA polymerase (reverse transcriptase) to copy RNA into cDNA (complementary DNA) using deoxyribonucleoside triphosphates. When performing RT-PCR in a single container, buffers and cofactors must support the optimal activity of both reverse transcriptase and PCR enzyme.

[0055] The term "identifier sequence" refers to a nucleotide sequence that can be used to a) identify and / or trace the source of polynucleotides in a reaction, b) count the number of times an initial molecule is sequenced, and c) pair with sequence reads from different strands of the same molecule.

[0056] The term "adaptor" refers to a nucleic acid attached to a polynucleotide, polynucleotide target, or target amplicon during sequencing preparation. Adaptors can be attached using primer extension, ligation, or other techniques. Adaptors can be single-stranded or double-stranded and can contain DNA, RNA, and / or artificial nucleotides. Adaptors can be located at the ends of polynucleotides, or they can be located in the middle or inner portions. Adaptors can add one or more functional regions to the polynucleotide, such as providing primer-binding sites for later stages of primer extension or sequencing, or providing a marker sequence. For example, an adaptor can contain universal primers and / or universal initiation sites, including initiation sites for sequencing and / or capture sites for NGS sequencing systems.

[0057] The term "polynucleotide target" refers to the polynucleotide of interest. An isolated polynucleotide target molecule is a single molecule present in a composition that does not contain other polynucleotide target molecules.

[0058] The term "region" refers to a nucleotide sequence that can be single-stranded or double-stranded.

[0059] Other definitions of terms may appear throughout the specification or be understood based on the specification.

[0060] The precise nucleotide sequences of target-specific primers and universal primers are generally not important to this technique and can be selected by the user based on the teachings of this disclosure.

[0061] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0062] All patents and publications mentioned in this article, including all sequences disclosed in such patents and publications, are expressly incorporated by reference.

[0063] In one aspect, this disclosure provides a method for performing a multi-stage primer extension reaction in a closed container by preparing a primer extension mixture in the container, wherein the mixture comprises: i) a polynucleotide target; ii) an unblocked primer; and iii) a blocked primer. The mixture will typically contain other primer extension agents, such as deoxyribonucleotide triphosphates (dNTPs), DNA polymerase or other primer extension enzymes, and a buffer. In some embodiments, the unblocked or blocked primer comprises a target-specific primer. The target-specific primer comprises a 5' region and a 3' region, wherein the 3' region contains a target-specific sequence and the 5' region contains a universal sequence. The universal primer comprises a universal sequence or its complementary sequence, and an optically cleavable blocking group at its 3' end. In some embodiments, the container is sealed after preparing the mixture, and an early polymerase chain reaction is performed with the mixture to produce a target amplicon or target cDNA. The blocked primer can be unblocked in the mixture to produce an unblocked primer containing the universal sequence or its complementary sequence. In some embodiments, unblocking is performed without opening the container. Late-stage primer extension reactions can be performed using unblocked primers and target amplicon or target cDNA, wherein the unblocked primers amplify the target amplicon. In some embodiments, unblocking and / or late-stage primer extension reactions are performed by photocleaving the blocking group from a blocked universal primer, for example by exposing the blocking primer in a sealed container to ultraviolet light.

[0064] In another aspect, this technology relates to novel compositions for performing primer extension reactions, wherein the compositions comprise a) a polynucleotide target; b) primers for unblocking in an early stage; and c) primers for blocking in a later stage. In some aspects, the target-specific primers comprise a 5' region and a 3' region, wherein the 3' region contains a target-specific sequence and the 5' region contains a universal sequence. In other aspects, the universal primers comprise a universal sequence or a portion thereof, and a photocleavable blocking group. In some aspects, the compositions are prepared in a container that is sealed after the composition is prepared. In other aspects, the universal primers are unblocked by exposure to ultraviolet light or other photocleaving techniques.

[0065] In other aspects, this technology relates to a kit for practicing the methods of the present invention as described above. In some embodiments, the kit may comprise a composition for a multi-stage primer extension reaction as described above. In some embodiments, the kit may comprise a mixture containing unblocking primers (e.g., target-specific primers or reverse transcriptase primers) and blocking primers (e.g., universal primers or target-specific primers). In some embodiments, the kit includes a container containing a mixture of unblocking target-specific primers and blocking universal primers.

[0066] In some embodiments, the kit includes a container containing a mixture of unblocked RT primers and blocked target-specific primers.

[0067] In some embodiments of the methods and compositions of the present invention, the early-stage primers are present at a concentration in the range of 0.01 μM to 0.5 μM, and the late-stage primers are present at a concentration in the range of 0.2 μM to 1 μM. In some embodiments of multiplex PCR, the target-specific primers are present at a concentration in the range of 0.01 μM to 0.5 μM, and the universal primers are present at a concentration in the range of 0.2 μM to 1 μM. In some embodiments of RT-PCR, the RT primers are present at a concentration in the range of 0.01 μM to 0.5 μM, and the blocked target-specific primers are present at a concentration in the range of 0.2 μM to 1 μM.

[0068] The compositions, methods, and kits described herein can be used to perform multi-stage primer extension reactions on polynucleotide targets (e.g., genomic DNA; mitochondrial DNA, messenger RNA, microRNA). Polynucleotide targets can be obtained from virtually any organism (including but not limited to plants, animals (e.g., reptiles, mammals, insects, worms, fish, etc.), tissue samples, bacteria, fungi (e.g., yeast), bacteriophages, viruses, cadaver tissue, archaeological / ancient samples, etc.). In some embodiments, the sample may contain polynucleotide targets derived from mammalian cells (e.g., human, mouse, rat, or monkey cells). Samples can be obtained from cultured cells or clinical sample cells (e.g., tissue biopsy, scraping, or irrigation) or forensic sample cells (e.g., cells from samples collected at crime scenes). In some embodiments, polynucleotide targets can be obtained from biological samples (e.g., cells, tissues, body fluids, and feces). Body fluids of interest include, but are not limited to, blood, serum, plasma, saliva, mucus, phlegm, cerebrospinal fluid, pleural fluid, tears, mammary duct fluid, lymph, sputum, synovial fluid, urine, amniotic fluid, and semen. In certain implementations, bodily fluids may be obtained from a subject, such as a human.

[0069] In some embodiments, the polynucleotide target comprises DNA or RNA obtained from clinical samples, such as those from patients who have or are suspected of having a disease or condition (e.g., cancer, inflammatory disease) or who are pregnant. In some embodiments, the sample can be prepared by extracting the polynucleotide target from archived patient samples (e.g., formalin-fixed paraffin-embedded tissue samples). In some embodiments, the patient sample may be a cell-free circulating DNA sample from bodily fluids (e.g., peripheral blood). In some embodiments, the polynucleotide target used in the early stages of the method of the invention is unamplified DNA that has not been denatured beforehand. In other embodiments, the polynucleotide target in the sample may have been partially fragmented (e.g., as in the case of FFPE samples and circulating cell-free DNA (cfDNA) (e.g., ctDNA)). In some embodiments, the compositions, methods, and kits can be used to perform multistage RT-PCR on polynucleotide targets derived from RNA, including polyA-graded mRNA, from virtually any organism or sample type.

[0070] Late-stage primers containing closed 3' ends

[0071] In some implementations, the late-stage primers are compounds according to Formula I:

[0072]

[0073] R1 is H or OH.

[0074] The bases in Formula I are cytosine, uracil, thymine, adenine, or guanine, or their modified pyrimidine and purine derivatives. The bases may be any substituted or unsubstituted nitrogen-containing parent heteroaromatic ring of the type commonly found in nucleic acids and their natural, substituted, modified, or engineered variants or analogues, capable of forming Watson-Crick and / or Husstan hydrogen bonds with appropriately complementary bases.

[0075] In Formula I, the cleavable terminating portion is a group that imparts polymerase-terminating properties to the compound. In some embodiments, the cleavable terminating portion is a portion of the following formula or its salt, tautomer, or optical isomer:

[0076]

[0077] Wherein R3 is an alkyl (C≤8) or a substituted alkyl (C1-8); R4 is hydrogen, hydroxyl, halogen, amino, nitro, cyano, azide, or mercapto; alkyl (C≤6), acyl (C≤6), alkoxy (C≤6), acyloxy (C≤6), alkylamino (C≤6), dialkylamino (C≤6), amide (C≤6), or any substituted form of these groups; R5 and R6 are each independently: hydrogen, hydroxyl, halogen, amino, nitro, cyano, azide, or mercapto; alkyl (C≤6), alkenyl (C≤6), alkynyl (C≤6), aryl (C≤6), aralkyl (C≤8), heteroaryl (C≤6), acyl (C≤6), alkoxy (C≤6), acyloxy (C≤6), alkylamino (C≤6), dialkylamino (C≤6), amide (C≤6), or any substituted form of these groups; the following groups:

[0078]

[0079] Where X is -O-, -S-, or -NH-; or alkyldiyl (C≤12), alkenyl (C≤12), alkynyl (C≤12), or any substituted form of these groups; Y is -O-, -NH-, alkyldiyl (C≤12) or a substituted alkyldiyl (C≤12); n is an integer from 0 to 6; and m is an integer from 0 to 6; or a connector-reporter molecule.

[0080] The optional linker in Formula I is one or more divalent groups, which serve as molecular bridges for covalent bonding between two other groups, such as -C(O)NH-, -C(O)O-, -NH-, -S-, -S(O)n, where n is 0, 1, or 2, -O-, -OP(O)(OH)O-, -OP(O)(O -O-, alkyldiyl, alkenyl, ynyl, aromatic alkenyl, heteroaromatic diyl, and combinations thereof. Some linkers have side-attached side chains or side-attached functional groups (or both). Optional reporter molecules are chemical moieties capable of generating a detectable signal directly or indirectly. Examples of reporter molecules include fluorescent dye groups, radiolabeled groups, or groups that affect the signal by chemiluminescence or bioluminescence. In some embodiments, the reporter molecule is selected from the group consisting of: xanthan, fluorescein, rhodamine, fluoroboron dipyrrole, anthocyanin, coumarin, pyrene, phthalocyanine, phycobiliproteins, and derivatives thereof.

[0081] The primer in Formula I is an oligonucleotide capable of forming a double strand with a polynucleotide target. In some embodiments, the primer is 8 to 100 nucleotides in length, or 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, or 25 to 40 nucleotides in length, or another length within another range disclosed herein.

[0082] In some embodiments, the universal primer comprises a 3' terminal nucleotide selected from the group consisting of: (a) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-uridine, (b) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-adenosine, (c) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-adenosine, (d) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropoxy]methyl-2'-deoxy-cytidine, (e) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropoxy]methyl-2'-deoxy-thymidine, and mixtures thereof, wherein the nucleosides are optionally substituted with linkers and / or reporter molecules. Exemplary mixtures include mixtures of the following substances: nucleotides (a) and (b); nucleotides (a), (b) and (c); nucleotides (a), (b), (c) and (d); nucleotides (a), (b), (c), (d) and (e); nucleotides (b) and (c); nucleotides (b), (c) and (d); nucleotides (b), (c), (d) and (e); nucleotides (a) and (c); nucleotides (a) and (d); nucleotides (a) and (e); nucleotides (a), (b) and (d); nucleosides Acids (a), (c), and (d); nucleotides (a), (c), (d), and (e); nucleotides (a), (b), (d), and (e); nucleotides (a), (b), (c), and (e); nucleotides (b) and (d); nucleotides (c) and (d); nucleotides (b) and (e); nucleotides (c) and (e); nucleotides (b), (c), and (e); nucleotides (b), (d), and (e); nucleotides (c), (d), and (e); nucleotides (d) and (e); and any other mixtures.

[0083] Methods, compositions, and kits for multiplex and multistage PCR

[0084] In another aspect, this disclosure provides methods and compositions for improving the efficiency of multiplex nucleic acid amplification. This disclosure also relates to reagents and methods for improving the efficiency of multi-stage nucleic acid amplification, particularly those designed to perform two or more amplification reactions sequentially within the same reaction mixture or container. Specifically, compositions with reduced primer dimer formation and aberrant amplification product formation are provided. Before UV unblocking, the blocked primers do not form any extendable double strands. After UV unblocking, they become primers capable of extension. Such primers are particularly useful when early and late amplification reactions occur in a single reaction mixture or container. Additional information relating to multiplex and multi-stage PCR amplification reactions and related reagents is contained in WO2018 / 10842A1, the entire contents of which are incorporated herein by reference.

[0085] In another aspect, this technique relates to multi-stage RT-PCR using unblocked RT primers as early-stage primers and blocked primers as late-stage primers. For example, the late-stage primers may contain target-specific primers having a light-cleavable blocking group at their 3' end. In some embodiments, the polynucleotide target is reverse transcribed in the early stage using an unblocked RT primer via reverse transcriptase to produce target cDNA. Examples of RT primers include oligomeric (dT) primers, random polymers (N6-Nn, where n can be an integer, such as 7, 8, 9, or 10), or target-specific RT primers. The target cDNA is then amplified during late-stage PCR, for example, by light-cleaving of the unblocked target-specific primers that have been light-cleaved by the blocking group.

[0086] This technique specifically relates to multiplex nucleic acid amplification, in which two or more target sequences are amplified in parallel. This is typically achieved by including more than one pair of polynucleotide target-specific primers in a single nucleic acid amplification reaction.

[0087] This technique also involves multi-stage nucleic acid amplification, in which two or more distinct amplification reactions occur. Typically, the early amplification reaction utilizes target-specific primers for amplifying polynucleotide target molecules. These target-specific primers contain a 5' region and a 3' region, wherein the 3' region contains a target-specific sequence and the 5' region contains a universal sequence. As the reaction proceeds, the universal sequence is incorporated into the amplification product. In the later amplification reactions, universal primers containing a universal sequence or a portion thereof sufficient to hybridize with a complementary sequence to the universal sequence are used to amplify the amplification product from the early amplification.

[0088] The method of the present invention will typically include multiple primer extension cycles within each stage. For example, an early stage may include at least 3, 4, 5, 6, 7, 8, 9, 10 or more primer extension cycles, and / or up to 20, 18, 16, 14, 12 or fewer PCR cycles. Similarly, a late stage may include at least 3, 4, 5, 6, 7, 8, 9, 10 or more primer extension cycles and / or up to 20, 18, 16, 14, 12 or fewer primer extension cycles. The method of the present invention may also include additional primer extension stages before or after the early and / or late stages. For example, a primer extension stage may precede the early stage to provide a higher quantity of input polynucleotides for the early stage, and a PCR stage may follow the late stage to provide a higher quantity of output polynucleotides for sequencing or other applications.

[0089] In some implementations, this late-stage amplification involves universal primers incorporating additional sequences potentially needed for further downstream processing and identification. Therefore, universal amplification is governed by the fact that late-stage amplification is performed independently of the specific target sequence of the initial target molecule being amplified. Universal amplification relies on incorporating additional sequences (such as universal sequences as described herein) into the amplification product from the early amplification reaction, which can act as primer-binding sites in late-stage amplification. Thus, the primer region of the primer in late-stage amplification corresponds to the universal sequence. Primers containing such primer regions are referred to herein as "universal primers".

[0090] The universal primers of this technique contain an optically cleavable blocking group at their 3' ends. The blocked universal primers are inactive for PCR amplification, even if they are present during the PCR amplification phase. Because the 3' blocking group of this technique is optically cleavable, it can be removed by exposing the blocked universal primers to ultraviolet light or other photocleaving techniques. Ultraviolet light exposure removes the blocking group and produces universal primers active for PCR amplification. Therefore, the universal primers of this technique can be present, but are blocked and substantially inactive during the early stages of target-specific PCR amplification, and then activated by exposure to ultraviolet light before the later stages of universal PCR amplification.

[0091] The polynucleotide targets to be amplified using this technique are generally unrestricted. The reagents and methods used in this technique can amplify any suitable polynucleotide target molecule. Multiple different polynucleotide target molecules can be targeted. This may involve using multiple polynucleotide target-specific primer pairs. Therefore, the term polynucleotide target generally refers to the desired sequence of the nucleic acid molecule to be amplified, whether as part of the initial polynucleotide target molecule present before amplification begins or as a polynucleotide target amplicon molecule generated during amplification.

[0092] Polynucleotide targets are molecules that contain or are derived from DNA or RNA molecules. As mentioned above, RNA can be obtained from the same sample type as DNA. RNA can be messenger RNA (mRNA), microRNA (miRNA), etc. In some embodiments, reverse transcriptase is used to reverse transcribe RNA to form complementary DNA (cDNA) molecules, which can then be amplified using this technique.

[0093] The target-specific primer pairs of this technique are designed to amplify polynucleotide targets and typically incorporate a universal sequence. This universal sequence does not hybridize with the initial polynucleotide target molecule. This function is provided by the target-specific 3' region of the target-specific primers. However, once the universal sequence has been included in the amplification product, it (or its complementary sequence) can act as a primer-binding site for hybridization with the universal primer in later amplification steps.

[0094] According to some implementations, the late PCR stage for universal amplification can also be used to include one or more adaptor sequences in the late amplicons. The adaptor sequence can be any suitable sequence for downstream processing. Downstream processing allows for the detection and / or quantification of polynucleotide targets or their amplicons from a sample. For example, an adaptor sequence complementary to an oligonucleotide immobilized on a suitable solid surface allows for the immobilization of sequences incorporating such adaptors. Other applications rely on adaptors that hybridize with oligonucleotides in a liquid. The adaptor can be used for array-based or sequencing-based analyses. In some implementations, the adaptor sequence can be any suitable adaptor sequence for high-throughput nucleic acid sequencing. Such sequencing is typically and preferably performed using a next-generation sequencing (NGS) platform.

[0095] In some embodiments, the universal primer further includes one or more primer binding sites. For example, a first (or forward) universal primer may include a first primer binding site, and a second (or reverse) universal primer may include a second primer binding site, wherein the first and second primer binding sites are configured to bind to different primers (e.g., the first and second primer binding sites do not have substantially identical sequences but are substantially complementary). The first and / or second primer binding sites may be sequencing primer binding sites, capture primer binding sites, or combinations thereof. For example, the first universal primer may include a first flow cell amplification primer binding site, and the second universal primer may include a second flow cell amplification primer binding site. In some embodiments, when the first primer binding site is a sequencing primer binding site, the first universal primer further includes a marker sequence upstream of the first primer binding site. For example, the first universal primer may include a universal capture sequence upstream of the marker sequence. In some embodiments, when the second primer binding site is a sequencing primer binding site, the second universal primer further includes an index downstream of the second primer binding site. For example, the second universal primer may be contained in a universal capture sequence or its complementary sequence downstream of the identifier sequence. In some embodiments, when the first universal primer does not contain an index, the universal capture site is upstream of the first primer binding site.

[0096] In some embodiments, various primers of this technology can also be used to add one or more identifier sequences (also referred to as indexes or barcodes) to the amplification products. For some aspects of this technology involving target-specific primers used in early amplification, sample identifier sequences and / or molecular identifier sequences are advantageously included in the primers. In certain embodiments, the length of the identifier sequence can range from 2 to 36 nucleotides, or 6 to 30 nucleotides, or 8 to 20 nucleotides. In some embodiments, the identifier sequence may contain a “degenerate base region” or “DBR,” where the terms “degenerate base region” and “DBR” refer to a type of molecular identifier sequence whose complexity is sufficient to help distinguish fragments to which a DBR has been added.

[0097] The term "sample identifier sequence" refers to a class of identifier sequences that can be added to polynucleotides, wherein the sequence identifies the source of the polynucleotide (i.e., the sample from which the polynucleotide originates). In use, each sample is labeled with a different sample identifier sequence (e.g., a sequence is appended to each sample, with different samples appended to different sequences), and the labeled samples are merged. After sequencing the merged samples, the sample identifier sequence can be used to identify the source of the sequence. The term "molecular identifier sequence" refers to a class of identifier sequences that can be added to polynucleotides, wherein the sequence identifies an individual polynucleotide or its amplicons.

[0098] In some embodiments, the universal primer comprises a first universal primer and a second universal primer, wherein the first universal primer comprises, in 5' to 3' order: (i) a first adaptor sequence; (ii) a molecular identifier sequence; and (iii) a universal primer region that is identical (in the 5' to 3' direction) to at least a portion of the first universal sequence; and the second universal primer comprises, in 5' to 3' order: (i) a second adaptor sequence; (ii) a sample identifier sequence; and (iii) a universal primer region that is identical (in the 5' to 3' direction) to at least a portion of the second universal sequence.

[0099] In some embodiments, this technology provides a method for performing multiplex and multistage PCR reactions in a single reaction mixture. In some embodiments, all amplification steps (i.e., early and late PCR phases) from the polynucleotide target in the PCR mixture up to and including the production of a related amplification product containing a universal sequence are performed without the need for separation, removal, or addition of components. In some embodiments, it is not necessary to perform the target-specific amplification phase in a mixture without universal primers, or to add universal primers between the early and late phases, or to purify the early amplification product prior to universal amplification. In some embodiments, all PCR reagents required for the method (i.e., to produce further amplification products) are combined prior to the early amplification phase. Therefore, the method can be performed in a single reaction vessel, and the vessel does not need to be opened after adding all PCR reaction mixture components. Once the reaction mixture has been formed (except for performing the amplification itself, such as thermal cycling) until the universal amplification product has been produced, no further manipulation or opening of the reaction vessel is required. Therefore, the method of the present invention can be considered a “closed-vessel” method. The method of the present invention is highly advantageous because the user does not need to add universal primers between the early and late phases.

[0100] In some embodiments, all primer extension phases (i.e., both early and late phase primer extension phases) from the polynucleotide target in the mixture up to and including the generation of a related target amplification product containing a universal sequence are performed without the need for separation, removal, or addition of components. In some embodiments, it is not necessary to perform the target-specific amplification phase in the mixture without universal primers, or to add universal primers between the early and late phases, or to purify the early target amplification product prior to universal amplification. In some embodiments, all primer extension reagents required for the method (i.e., to generate further amplification products) are combined before performing the early amplification phase. Therefore, the method of the present invention can be performed in a single reaction vessel, and the vessel does not need to be opened after all components of the reaction mixture have been added. Once the reaction mixture has been formed (except for performing the amplification itself, such as thermal cycling) until the universal amplification product has been generated, no further manipulation or opening of the reaction vessel is required. Therefore, the method of the present invention can be considered a “closed-vessel” method. The method of the present invention is highly advantageous because the user does not need to add late-phase primers between the early and late phases.

[0101] The method of this invention also encompasses performing additional steps after the generation of a universal amplification product (i.e., after universal amplification or a later stage amplification). Such methods are not limited to the same reaction mixture or reaction vessel. These methods may involve the detection and, optionally, quantification of polynucleotide target molecules. In some embodiments, the method of this technique is used to identify and, optionally, quantify specific polynucleotide target molecules. In other embodiments, the method further includes sequencing additional amplification products. Sequencing is typically performed in massively parallel mode, for example, by using next-generation sequencing (NGS) technology. Sequencing may be performed in a reaction mixture different from the amplification reaction of this technique.

[0102] This technique also relates to a multi-stage RT-PCR reaction in which two or more distinct amplification reactions occur in a single container. cDNA synthesis is performed independently of PCR by blocking the 3' ends of target-specific PCR primers with light-cleavable blocking groups. cDNA synthesis is performed at a constant temperature optimal for reverse transcriptase and is unaffected by PCR primers, which would otherwise interact nonspecifically to produce primer dimers and other artificial products. Therefore, the target-specific PCR primers of this technique can be present but are substantially inactive during cDNA synthesis and then activated by exposure to UV light prior to a later-stage primer extension reaction (e.g., PCR). As described above for multi-stage mPCR reactions, the RT-PCR method can be performed in a single container without further manipulation.

[0103] This disclosure also provides kits for practicing the methods of the present invention as described herein. In some embodiments, the kit may contain a composition for multi-stage PCR as described above. In some embodiments, the kit may contain a PCR mixture comprising target-specific primers and blocking universal primers having an optically cleavable blocking group at their 3' ends. The target-specific primers and blocking universal primers may be in a mixture in a single container. The kit of this technology may additionally contain suitable reagents (e.g., buffers, etc.) for performing multi-stage PCR. The various components of the kit may be present in separate containers, or, if necessary, certain compatible components may be pre-assembled into a single container. In addition to the reagents described above, the kit may also contain any additional components used in the methods described above, such as one or more enzymes and / or buffers, etc.

[0104] In some embodiments, the kit may contain a composition for multi-stage RT-PCR as described above. In some embodiments, the kit may contain an RT-PCR mixture comprising cDNA synthesis primers (oligomeric (dT) or random polymers) and target-specific primers having a lightly cleavable blocking group at their 3' ends. The cDNA synthesis and blocking target-specific primers may be in a mixture in a single container. The kit of this technology may additionally contain suitable reagents for performing multi-stage RT-PCR, which may be provided in any of the forms described above.

[0105] In addition to the components described above, the kit may also include instructions for use in practicing the method of the present invention using the components of the kit, i.e., instructions for performing multi-stage amplification of polynucleotide targets. Instructions for use in practicing the method of the present invention may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic. Therefore, the instructions may exist as a packaging insert in the kit, in a label on the kit container or its components (i.e., associated with the packaging or sub-packaging), etc. In other embodiments, the instructions exist as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, floppy disk, etc. In other embodiments, no actual instructions for use are included in the kit, but a method for obtaining the instructions for use from a remote source (e.g., via the Internet) is provided. An example of this embodiment is a kit that includes a URL where the instructions for use can be viewed and / or downloaded. Like the instructions for use, this means of obtaining the instructions for use is recorded on a suitable substrate.

[0106] Optically cleavable blocking groups

[0107] This technology relates to primers that are reversibly blocked at their 3' ends by a photocleavable blocking group. These blocked primers may be present during an early-stage primer extension reaction, but are blocked from extending during this stage. After they are unblocked, they become capable of extending in a later-stage primer extension reaction. The photocleavable blocking group of this invention comprises a nucleotide attached to the 3' end of the later-stage primer, at which the nucleotide blocks PCR amplification. The blocked primers are substantially inactive for PCR amplification until they are unblocked and activated by exposure to ultraviolet light or other photocleaving techniques. Various photocleavable blocking groups can be included in later-stage primers, such as those described in U.S. Patent Nos. 8,969,535, 9,200,319, and 10,041,115, the entire contents of which are incorporated herein by reference. It is contemplated that the later-stage primers of this invention may contain any photocleavable blocking group at their 3' ends such that the later-stage primers are substantially inactive for PCR amplification prior to being unblocked. In some embodiments, the optically cleavable blocking group has a blocking efficiency of about 90% to about 100%.

[0108] The photocleavable blocking group is designed to reversibly block and terminate DNA synthesis, and is then efficiently cleaved by exposure to ultraviolet light, thereby activating the primer. In some embodiments, the photocleavable blocking group is in the form of a nucleotide compound containing adenine, cytosine, guanine, thymine, uracil, or modified pyrimidine and purine derivatives thereof, such as 7-hydroxy-7-deadenine / guanine. In other embodiments, the cleavable group can be derivatized to include a reporter molecule, such as a dye. In some embodiments, adenine, cytosine, guanine, thymine, uracil, or modified pyrimidine and purine derivatives thereof can be covalently attached to the photocleavable protecting group, such as 2-nitrobenzyl. In some embodiments, the 2-nitrobenzyl is derivatized to enhance its termination of DNA synthesis. In some embodiments, the photocleavable protecting group, such as 2-nitrobenzyl, can also be derivatized with a fluorescent dye by covalently linking it to the photocleavable protecting group.

[0109] In some embodiments, the photocleavable blocking group comprises a nucleoside base covalently attached to a 2-nitrobenzyl group, and the α-carbon position of the 2-nitrobenzyl group is optionally substituted with an alkyl or aryl group. In other embodiments, the 2-nitrobenzyl group is functionalized to enhance termination and blocking properties as well as the photocatalytic deprotection rate. In other embodiments, the termination and blocking properties of the 2-nitrobenzyl group attached to the base and the α-carbon-substituted 2-nitrobenzyl group even occur when the 3'-OH group on the ribose is unblocked. In some embodiments, a photocleavable blocking group that is well tolerated by many commercially available DNA polymerases is selected. In some embodiments, the α-carbon-substituted 2-nitrobenzyl group may also be derivatized to include a selected fluorescent dye or other reporter molecule.

[0110] Methods for preparing optically cleavable blocking groups

[0111] The photocleavable blocking group is in the form of a nucleotide compound containing a photocleavable protecting group designed to terminate DNA synthesis and facilitate rapid cleavage. These are combined with a primer precursor and added to the 3' end of the precursor, for example, by annealing the primer precursor with a template using DNA polymerase, or alternatively by template-independent single-base extension of the primer precursor using terminal deoxynucleotide transferase (TdT). Therefore, universal primers containing the photocleavable blocking group are inactive for further extension.

[0112] In another embodiment, the nucleotide containing the optically cleavable blocking group is a compound according to the following formula, which can be attached to the 3' end of a universal primer:

[0113]

[0114] Where R1 is H or OH, R2 is H, monophosphate, diphosphate, triphosphate, or α-thiotriphosphate, the base is cytosine, uracil, thymine, adenine, or guanine, or a modified pyrimidine or purine derivative thereof, the cleavable termination part is a group that imparts polymerase-terminating properties to the compound, and the optional linker is a bifunctional group. The base in Formula II is cytosine, uracil, thymine, adenine, or guanine, or a modified pyrimidine or purine derivative thereof. As mentioned above, the base can be any substituted or unsubstituted nitrogen-containing parent heteroaromatic ring of the type commonly found in nucleic acids and their natural, substituted, modified, or engineered variants or analogues, capable of forming Watson-Crick and / or Husstein hydrogen bonds with appropriately complementary bases.

[0115] In Formula II, the cleavable terminator is a group that imparts the polymerase-terminating property to the compound. The optional linker in Formula I is one or more divalent groups that act as a molecular bridge covalently bonded between two other groups. The optional reporter molecule is a chemical moiety capable of generating a detectable signal directly or indirectly. Examples of cleavable terminators, optional linkers, and optional reporter molecules are described above with respect to Formula I, and those illustrative cleavable terminators, optional linkers, and optional reporter molecules may also be incorporated into Formula II.

[0116] Example

[0117] Example 1: Production of optically cleavable 3' blocking primers

[0118] In this embodiment, primers with a blocking group at their 3' ends are synthesized. Optically cleavable blocking primers are produced by performing a single-base extension of the primer precursor. The primer precursor (Numb1-1) is annealed with a DNA template, and a nucleotide containing the optically cleavable blocking group (LT-dG) is incorporated into the 3' end of the primer precursor by a single-base extension. The product is purified and analyzed by reverse-phase high-performance liquid chromatography (HPLC). Figure 2 The leftmost peak is shown as the product of primers and templates annealed before the addition of LT-dG, the middle peak is shown as the single-base extension product of primers with added LT-dG, and the rightmost peak is the excess and unincorporated LT-dG.

[0119] Example 2: Optically cleavable blocking primers can be deblocked by ultraviolet light.

[0120] In this embodiment, the ability to understand primers that have a blocking group at their 3' end is evaluated. Figure 4 The HPLC traces are shown, illustrating the HPLC-purified universal primer with a photocleavable blocking group at the 3' end (main peak on the right) and the universal primer after 10 seconds of exposure to 365 nm UV light (peak on the left). The increased HPLC mobility is due to the cleavage of the photocleavable blocking group from the 3' end of the primer by UV light. This demonstrates that the photocleavable blocking primers of this technique can be effectively unblocked by UV light and then extended by DNA polymerase.

[0121] Example 3: Only after exposure to ultraviolet light can light-cleavable blocking primers be extended in PCR.

[0122] In this embodiment, the use of blocking primers for PCR amplification was evaluated. Figure 5Images of three PCR products are shown using a Bioanalyzer 2100. The lane labeled "PCR using unblocked primers" represents the positive control, showing the amplification product of a 305 bp gDNA fragment using unblocked primers (Numb1-FP and Numb1-RP). The lane labeled "PCR using blocking primers" represents an attempt at PCR using an unblocked reverse primer (Numb1-RP) and a lightly cleavable blocking forward primer (Numb1-F*). This lane shows minimal PCR amplification because the blocking primers cannot extend during PCR amplification. The lane labeled "PCR using blocking primers exposed to UV" represents PCR using unblocked reverse primers and lightly cleavable blocking forward primers after the blocking group on the forward primer (Numb1-F*) has been cleaved by exposure to UV light. This lane shows amplification of the PCR product because the forward primers are unblocked and become able to extend during PCR amplification. These results indicate that the blocking primers of this technique do not extend during PCR amplification, but can be unblocked and activated by exposure to ultraviolet light to extend during PCR amplification.

[0123] Example 4: Only after exposure to ultraviolet light can light-cleavable blocking primers be extended in RT-PCR.

[0124] In this embodiment, the use of blocked target-specific primers was evaluated in RT-PCR as another implementation of a multi-stage primer extension reaction. Figure 6 shows Bioanalyzer 2100 images of products from a single-container RT-PCR reaction performed with either a lightly cleavable blocked β-actin reverse primer (inset A; R*) or a lightly cleavable blocked Numb1 forward primer (inset B; F*). The blocked tubes were exposed to UV for up to 3 minutes between cDNA synthesis and thermal cycling. Without UV exposure, no target-specific products were generated in either assay, indicating that β-actin R* and Numb1 F* remained inactive during both cDNA synthesis and the PCR step. For β-actin, an additional control showed that reverse transcription was initiated from β-actin R* during the period between UV exposure and the initial PCR denaturation step (not shown). Non-specific interactions can be prevented during this short timeframe by performing UV exposure at high temperatures. The results showed that the blocked target-specific primers extended only in RT-PCR after exposure to ultraviolet light.

[0125] Exemplary Implementation

[0126] The exemplary implementations provided based on the currently disclosed subject matter include, but are not limited to, the following:

[0127] Implementation Method 1. A method for performing a multi-stage primer extension reaction in a sealed container. The method includes a) preparing a primer extension mixture in the container, wherein the mixture contains i) a polynucleotide target; ii) an early-stage primer capable of primer extension; iii) a late-stage primer, the late-stage primer containing a lightly cleavable blocking group at its 3' end; iv) a primer extension enzyme; and v) a primer extension reagent. The container is sealed after the mixture is prepared. The method further includes b) performing an early-stage primer extension reaction with the early-stage primer to generate a target amplicon or target cDNA. The method includes c) unblocking the late-stage primer to generate unblocked late-stage primers, wherein the unblocking step is performed without opening the container. The method further includes d) performing a late-stage primer extension reaction with the unblocked late-stage primers and the target amplicon or target cDNA. The unblocked late-stage primers hybridize with and extend the target amplicon or target cDNA.

[0128] Implementation 2. According to the method of Implementation 1, the early stage primer includes a target-specific primer containing a 5' region and a 3' region, wherein the 3' region contains a target-specific sequence and the 5' region contains a universal sequence.

[0129] Implementation 3. According to the method of Implementation 2, wherein the late-stage primer comprises a universal primer, and the universal primer comprises the universal sequence or a portion thereof.

[0130] Implementation 4. The method according to any one of Implementations 1 to 3, wherein the early stage primers comprise reverse transcriptase (RT) primers.

[0131] Implementation 5. The method according to Implementation 4, wherein the late-stage primers include target-specific primers.

[0132] Implementation 6. The method according to any one of Implementations 1 to 5, wherein the unblocking step (c) includes exposing the late-stage primer in the sealed container to ultraviolet light.

[0133] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the blocking primer is a compound according to Formula I:

[0134]

[0135]

[0136] Wherein R1 is H or OH; the base is cytosine, uracil, thymine, adenine, or guanine, or a modified pyrimidine or purine derivative thereof; the cleavable termination part is a group that imparts polymerase-terminating properties to the compound; the optional linker is a divalent group; the optional reporter molecule is a chemical moiety capable of generating a detectable signal directly or indirectly; and the primer is an oligonucleotide capable of forming a double strand with a polynucleotide target.

[0137] Implementation Method 8. The method according to Implementation Method 7, wherein the severable termination portion is a portion of the following formula or a salt, tautomer, or optical isomer thereof:

[0138]

[0139] in:

[0140] R3 is an alkyl group (C≤8) or a substituted alkyl group (C1-8);

[0141] R4 is hydrogen, hydroxyl, halogen, amino, nitro, cyano, azide or mercapto; alkyl (C≤6), acyl (C≤6), alkoxy (C≤6), acyloxy (C≤6), alkylamino (C≤6), dialkylamino (C≤6), acylamino (C≤6), or any substituted form of these groups;

[0142] R5 and R6 are each independently: hydrogen, hydroxyl, halogen, amino, nitro, cyano, azide, or mercapto; alkyl (C≤6), alkenyl (C≤6), alkynyl (C≤6), aryl (C≤6), aralkyl (C≤8), heteroaryl (C≤6), acyl (C≤6), alkoxy (C≤6), acyloxy (C≤6), alkylamino (C≤6), dialkylamino (C≤6), acylamino (C≤6), or any substituted form of these groups; groups of the following formula:

[0143]

[0144] X is -O-, -S-, or -NH-; or alkyldiyl (C≤12), alkenyl (C≤12), ynyldiyl (C≤12), or any substituted form of these groups;

[0145] Y is -O-, -NH-, alkyldiyl (C≤12), or a substituted alkyldiyl (C≤12); n is an integer from 0 to 6; and

[0146] m is an integer between 0 and 6; or a connector-reporter molecule.

[0147] Embodiment 9. The method according to Embodiment 7, wherein the cleavable termination portion comprises a 2-nitrobenzyl substituent.

[0148] Embodiment 10. The method according to any one of Embodiments 7 to 9, wherein the primer is selected from oligonucleotides with a length between 8 and 100 nucleotides.

[0149] Embodiment 11. The method according to any one of Embodiments 7 to 10, wherein the base is selected from the group consisting of: adenine, cytosine, guanine, thymine, uracil, their modified pyrimidine and purine derivatives, and mixtures thereof.

[0150] Embodiment 12. A composition for performing a multi-stage primer extension reaction, the composition comprising a) a polynucleotide target; b) an early-stage primer capable of primer extension; and c) a late-stage primer, the late-stage primer comprising a light-cleavable blocking group at the 3' end, wherein the composition is prepared in a sealed container.

[0151] Embodiment 13. The composition of claim 12, wherein the late-stage primer is configured to be deblocked by exposure to ultraviolet light.

[0152] Embodiment 14. The composition according to any one of Embodiments 12 to 13, wherein the optically cleavable blocking group has a blocking efficiency of about 90% to about 100%.

[0153] Example 15. The composition according to any one of Examples 12 to 14, wherein the composition comprises at least 5 pairs of target-specific primers, or at least 10 pairs, or at least 20 pairs, or at least 50 pairs, or at least 100 pairs, or at least 200 pairs, or at least 500 pairs, or at least 1,000 pairs, or at least 2,000 pairs, or at least 5,000 pairs, or at least 10,000 pairs, or at least 20,000 pairs of target-specific primers.

[0154] Example 16. The composition according to any one of Examples 12 to 15, wherein the early-stage primer is present at a concentration of 0.01 μM to 0.5 μM, and the late-stage primer is present at a concentration of 0.2 μM to 1 μM.

[0155] Embodiment 17. The composition according to any one of Embodiments 12 to 16, wherein the late-stage primer comprises a 3' terminal nucleotide selected from the group consisting of:

[0156] 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-uridine,

[0157] 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-adenosine,

[0158] 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-guanosine,

[0159] 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-cytidine,

[0160] 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-thymidine,

[0161] And mixtures thereof, including any two, three, four or five of the aforementioned nucleotides.

[0162] Implementation 18. A method for preparing a light-cleavable blocking primer, the method comprising: a) providing a primer precursor having a 3' end; and b) i) forming a double strand of the primer precursor hybridizing with a template, wherein the template has a 5' overhang of at least one nucleotide relative to the 3' end of the primer precursor; and extending the primer precursor at its 3' end by incorporating a nucleotide containing a light-cleavable blocking group with a DNA polymerase; or ii) extending the primer precursor at its 3' end by incorporating a nucleotide containing a light-cleavable blocking group with a template-independent DNA polymerase.

[0163] Embodiment 19. The method of claim 18, wherein the nucleotide comprising the optically cleavable blocking group is a compound of formula II:

[0164]

[0165] Wherein R1 is H or OH; R2 is H, monophosphate, diphosphate, triphosphate, or α-thiotriphosphate; the base is cytosine, uracil, thymine, adenine, or guanine, or a modified pyrimidine or purine derivative thereof; the cleavable termination part is a group that imparts the polymerase-terminating property to the compound; the optional linker is a divalent group; and the optional reporter molecule is a chemical part that can directly or indirectly generate a detectable signal.

Claims

1. A method for performing a multi-stage primer extension reaction in a closed container, the method comprising: a) Prepare a primer extension mixture in a container, wherein the mixture comprises: i) Polynucleotide targets; ii) Primers capable of early-stage primer extension; iii) Late-stage primers, wherein the late-stage primers contain an optically cleavable blocking group at the 3' end; iv) Primer elongase; as well as v) Primer extension reagent, The container is sealed after the mixture is prepared. b) Perform an early-stage primer extension reaction using the early-stage primers to generate a target amplicon or target cDNA; wherein the early-stage primers include target-specific primers containing a 5' region and a 3' region, wherein the 3' region contains a target-specific sequence and the 5' region contains a universal sequence. c) Deblocking the late-stage primers to produce deblocked late-stage primers, wherein the deblocking step is performed without opening the container; and d) Perform a late-stage primer extension reaction using the unblocked late-stage primers and the target amplicon or target cDNA. The late-stage primers for unblocking hybridize with and extend the target amplicon or target cDNA.

2. The method of claim 1, wherein the late-stage primer comprises a universal primer, the universal primer comprising the universal sequence or a portion thereof.

3. The method according to claim 1, wherein the early stage primer comprises a reverse transcriptase primer.

4. The method of claim 3, wherein the late-stage primers comprise target-specific primers.

5. The method of claim 1, wherein the unsealing step (c) comprises exposing the late-stage primers in the sealed container to ultraviolet light.

6. The method according to claim 1, wherein the blocking primer is a compound according to formula I: Formula I in: R1 is H or OH; The bases are cytosine, uracil, thymine, adenine, or guanine, or their modified pyrimidine and purine derivatives; The cleavable termination portion is a group that imparts the polymerase-terminating property to the compound; The optional linker is a divalent group; The optional reporter molecule is a chemical moiety capable of generating a detectable signal directly or indirectly; and Primers are oligonucleotides that can form double strands with polynucleotide targets.

7. The method of claim 6, wherein the severable termination portion is a portion of the following formula or a salt, tautomer, or optical isomer thereof: in: R3 is a C≤8 alkyl or a substituted C1-8 alkyl; R4 is hydrogen, hydroxyl, halogen, amino, nitro, cyano, azide or mercapto; C≤6 alkyl, C≤6 acyl, C≤6 alkoxy, C≤6 acyloxy, C≤6 alkylamino, C≤6 dialkylamino, C≤6 acylamino, or any substituted form of these groups. R5 and R6 are each independently: hydrogen, hydroxyl, halogen, amino, nitro, cyano, azide, or mercapto; C≤6 alkyl, C≤6 alkenyl, C≤6 alkynyl, C≤6 aryl, C≤8 aralkyl, C≤6 heteroaryl, C≤6 acyl, C≤6 alkoxy, C≤6 acyloxy, C≤6 alkylamino, C≤6 dialkylamino, C≤6 acylamino, or any substituted form of these groups; groups of the following formula: or X is -O-, -S-, or -NH-; or C≤12 alkyldiyl, C≤12 olefinicdiyl, C≤12 alkynyldiyl, or any substituted form of these groups; Y is -O-, -NH-, C≤12 alkyldiyl or substituted C≤12 alkyldiyl; n is an integer from 0 to 6; and m is an integer between 0 and 6; or a connector-reporter molecule.

8. The method of claim 6, wherein the cleavable termination portion comprises a 2-nitrobenzyl substituent.

9. The method of claim 6, wherein the primer is selected from oligonucleotides with a length between 8 and 40 nucleotides.

10. The method of claim 6, wherein the base is selected from the group consisting of: adenine, cytosine, guanine, thymine, uracil, their modified pyrimidine and purine derivatives, and mixtures thereof.

11. The method according to claim 1, wherein the optically cleavable blocking group has a blocking efficiency of 90% to 100%.

12. The method of claim 1, wherein the early-stage primer is present at a concentration of 0.01 µM to 0.5 µM, and the late-stage primer is present at a concentration of 0.2 µM to 1 µM.

13. The method of claim 12, wherein the late-stage primer comprises a 3' terminal nucleotide selected from the group consisting of: 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-uridine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-adenosine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-guanosine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-cytidine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propoxy]methyl-2'-deoxy-thymidine, And their mixtures.

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