Compositions and methods for enhancing reverse transcriptase activity and / or reducing reverse transcriptase inhibition

Abstract

The present application relates to compositions and methods for enhancing reverse transcriptase (RT) activity and / or reducing inhibition of RT by inhibitors (e.g., formalin, tannic acid and / or heparin). In some embodiments, RT inhibition is reduced by adding potassium glutamate, histidine hydrochloride monohydrate, poloxamer 188, or any combination thereof to a reaction mixture comprising a polymerase. In other embodiments, RT is enhanced by adding sodium polyethylene sulfonate (PVSA) to the reaction mixture. The invention also provides oligonucleotide primers for reverse transcription of a target sequence and enhancement thereof. These primers have a high GC content or a low GC content. Also provided are methods of using an RT inhibition reducing agent or an RT enhancer in a composition having an RNA template and RT to improve RT yield, RT sensitivity, or RT tolerance to various chemicals.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 866,182, filed on June 25, 2019, which is incorporated herein by reference in its entirety.

[0003] The sequence listing for this application is referenced as "Seq-List.txt," which was created on June 20, 2020 and is 5KB in size. The entire contents of the sequence listing are incorporated herein by reference in their entirety. SUMMARY OF THE INVENTION

[0005] The inventors surprisingly found that in the presence of an oligonucleotide primer containing a randomized primer with high and / or low GC content, the activity of reverse transcriptase (RT) can be enhanced by synthesizing complementary DNA (cDNA) from an RNA template with high and low GC content. It is also surprisingly found that the synthesis of cDNA can be carried out by RT in the presence of an additive (e.g., potassium glutamate, histidine hydrochloride monohydrate and / or poloxamer 188) to reduce the inhibition of reverse transcriptase by heparin, formalin or tannic acid. It is also surprisingly found that the activity of reverse transcriptase (RT) can be enhanced by synthesizing complementary DNA (cDNA) from an RNA template in the presence of a salt of polyvinyl sulfonic acid (PVSA) (e.g., sodium salt of PVSA). A variety of reaction mixtures comprising additives disclosed herein are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 Effect of K-Glu (potassium glutamate) on heparin tolerance. Potassium glutamate improves the tolerance of RT to heparin. Heparin (0.003U / μl) causes a Cq delay of more than 7 in the RT reaction, while when 100mM K-Glu is present, the Cq delay is less than 1.

[0008] Figure 2 .K-Glu (potassium glutamate) improves heparin tolerance without affecting tolerance to other inhibitors. Potassium glutamate improves heparin tolerance without affecting tolerance to 7.5% ethanol and 50 mM guanidine hydrochloride. (NH4)2SO4 also improves heparin tolerance, although not as effectively as K-Glu, but it impairs ethanol and GC tolerance.

[0009] Figure 3 Effect of poloxamer on tannic acid tolerance. Poloxamer improves tannic acid tolerance. Tannic acid at 0.1 μg / μl in the RT reaction severely inhibited the RT process and caused a Cq delay of more than 13, while the presence of 0.5% poloxamer effectively improved the RT tolerance to tannic acid (less than 2 Cq delay).

[0010] Figure 4 . Effect of Histidine-HCl on Formalin Tolerance. Histidine-HCl improves formalin tolerance during RT reaction. In the absence of Histidine-HCl, the RT product has about 4Cq delay, while in the presence of 15 to 20 mM Histidine-Cl, there is less than 1Cq delay.

[0011] Figure 5 Effect of PVSA on RT yield and sensitivity. When RNA input is limited (reduced) in the RT reaction, PVSA can improve RT yield and make RNA detection more sensitive. For example, this paper shows that at 0.1 ng RNA input, PVSA can improve Cq (earlier than without PVSA) by about 3 cycles and increase sensitivity by about 10-fold. Summary of the invention

[0012] As used herein, the singular forms "a", "an", and "the" include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent that the terms "includes", "comprising", "having", "containing", "having", or their grammatical variations are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising". The transitional terms / phrases "comprising", "including", "containing", "consisting essentially of", "consisting essentially of", "consisting of", and "consisting of" (and any grammatical variations thereof) are used interchangeably.

[0013] The phrase "consisting essentially of" or "composed essentially of" means that a claim covers embodiments that include the specified materials or steps, as well as other materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0014] The term "about" or "approximately" means within the acceptable error range of a particular value determined by one of ordinary skill in the art, which will depend on how the value is measured or determined, i.e., the limits of the measurement system. The terms "about" and "approximately" are meant to cover a range of ±20%, ±10%, or ±5% of a given value. Thus, in the context of compositions containing a certain amount of an ingredient using the term "about" or "approximately," these compositions may contain the specified amount of ingredient with a variation (error range) of 0-10% (X ± 10%) around that value.

[0015] In the present disclosure, range is a shorthand form to avoid unnecessary detailed listing and description of each value in the range. Where appropriate, any suitable value in the range can be selected as the upper limit, lower limit or end point of the range. For example, the range 0.1-1.0 represents the final values ​​0.1 and 1.0, and the intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges are included in 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. It is envisioned that there are values ​​with at least two significant figures in a range, for example, the range of 5-10 represents all values ​​between 5.0 and 10.0 and between 5.00 and 10.00, including the end points.

[0016] The term "simultaneously" or "concurrently" as used in methods for synthesizing cDNA from an RNA template using RT refers to adding one or more components to the composition simultaneously or at two different time points no more than 3 minutes apart. The phrase "after or before" as applied to methods for synthesizing cDNA from template RNA refers to adding more than one composition at two different time points more than 3 minutes apart, e.g., about 5 minutes, 30 minutes, 1 hour, about 2 hours, about 5 hours, or even longer.

[0017] Various techniques are known for quantitative amplification of nucleic acids. These techniques include the use of 5' to 3' exonuclease assays, e.g., Taqman TM Probes (see, e.g., U.S. Pat. Nos. 5,210,015 and 5,487,972, Heid et al., Genome Res. 6:986-994, 1996; Holland et al., Proc. Nat'l Acad. Sci. USA 88:7276-7280, 1991; and Lee et al., Nuc. Acids Res. 21:3761-3766, 1993). Other methods employ one or more probe oligonucleotides that are constructed so that when one or more oligonucleotides are hybridized to a target nucleic acid, a fluorescence change is produced. For example, one such method involves a dual fluorophore method utilizing fluorescence resonance energy transfer (FRET), e.g., LightCycler TM Hybridization probes, in which two oligonucleotide probes anneal to an amplicon (e.g., U.S. Pat. No. 6,174,670). Other examples of labeled oligonucleotides constructed to emit a signal when bound to a nucleic acid or incorporated into an extension product include: Scorpions TM probes (eg, Whitcombe et al., Nature Biotechnology 17:804-807, 1999, and U.S. Pat. No. 6,326,145), Sunrise (or Ampliflour TM) primers (e.g., Nazarenko et al., Nuc. Acids Res. 25:2516-2521, 1997, and U.S. Pat. No. 6,117,635), LUX.TM. primers and Molecular Beacons TM Probes (eg, Tyagi et al., Nature Biotechnology 14:303-308, 1996 and US Pat. No. 5,989,823).

[0018] The present invention provides a method for amplifying a nucleic acid molecule. The method involves mixing an RNA template with a composition comprising a reverse transcriptase, an additive that reduces RT inhibition and / or increases RT efficiency as described herein, and / or a polymerase.

[0019] In RT-PCR, the reaction mixture (in an appropriate buffer) is first incubated at a temperature sufficient to allow the synthesis of a DNA molecule that is complementary to at least a portion of the RNA template. The components of the reverse transcription reaction mixture generally include: an RNA template and a DNA primer from which complementary DNA (cDNA) is produced; a nucleic acid polymerase that exhibits reverse transcriptase activity; and appropriate nucleotide building blocks (dNTPs) required for nucleic acid synthesis. For the purposes of the present invention, cDNA is defined as any DNA molecule whose nucleic acid sequence is complementary to an RNA molecule.

[0020] RNA template is defined as any RNA molecule used to provide a nucleic acid sequence from a cDNA molecule that can be synthesized. cDNA synthesis from an RNA template is typically accomplished by utilizing a nucleic acid polymerase that exhibits reverse transcriptase activity. For purposes of the present invention, reverse transcriptase activity refers to the ability of an enzyme to polymerize a cDNA molecule from an RNA template, and the term "reverse transcriptase" (RT) broadly refers to any enzyme that possesses reverse transcriptase activity. Reverse transcription typically occurs at a temperature ranging from about 20°C to about 75°C, preferably about 35°C to about 70°C.

[0021] After reverse transcription of the RNA template to produce a cDNA molecule, the cDNA is incubated (in a suitable buffer) under conditions sufficient to replicate the cDNA molecule. The reaction mixture may be the same as the previous reverse transcription reaction mixture, as used in coupled (also referred to as continuous or one-step) RT-PCR, or the reaction mixture may contain aliquots of the previous reverse transcription reaction mixture and may be further modified for nucleic acid replication, as used in non-coupled (or two-step) RT-PCR. The components of the replication reaction mixture typically include: a nucleic acid template (cDNA in this case); a DNA primer; a nucleic acid polymerase; and suitable nucleotide building blocks required for nucleic acid synthesis. Nucleic acid replication refers to the polymerization of a nucleic acid whose sequence is determined by another nucleic acid and is complementary to another nucleic acid. DNA replication, as used herein, is synonymous with DNA amplification. Preferably, DNA amplification occurs repeatedly, so that both chains of the nucleic acid sequence are replicated, i.e., the DNA is complementary to the RNA template, and the nucleic acid sequence of the DNA is substantially the same as the RNA template. Repeated or cyclic DNA replication can be advantageously accomplished using a thermostable polymerase in a polymerase chain reaction (PCR).

[0022] PCR is a well-known technique in the art. PCR is used to amplify nucleic acids by subjecting the reaction mixture to the following cycles: (i) nucleic acid denaturation, (ii) oligonucleotide primer annealing, and (iii) nucleic acid polymerization. Preferred amplification reaction conditions include thermal cycling, i.e., alternating the temperature of the reaction mixture to promote each step of the PCR cycle. PCR is typically extended by multiple cycles of denaturation, annealing, and replication, (optionally and preferably) enhanced by an initial extended denaturation step and a final extended extension (polymerization) step. Thermal cycling typically occurs in a temperature range of from about 23°C to about 100°C, preferably about 37°C to about 95°C. Nucleic acid denaturation typically occurs at about 90°C to about 100°C, preferably about 94°C. Annealing typically occurs at about 37°C to about 75°C, preferably about 60°C. Polymerization typically occurs at about 55°C to about 80°C, preferably about 72°C. The number of thermal cycles varies greatly, depending on the practitioner's preference, the number of DNA templates used, and the number of desired DNA products. Preferably, the number of PCR cycles is from about 5 to about 99, more preferably greater than about 20 cycles, and most preferably about 40 cycles.

[0023] Primers should be designed according to standard PCR guidelines with a length of 18 to 25 nucleotides and a GC content of 40% to 65%. Primer design should avoid internal secondary structures, as well as complementarity at the 3' end within each primer and primer pair. Optimal results may require titrating the concentration of primers between 100 and 500nM. A final concentration of 300nM per primer is effective for most reactions. In general, using equal concentrations of each primer can optimize reaction efficiency and / or specificity. For optimal results, the amplicon size used for quantitative PCR should be limited to 50-200bp.

[0024] The recommended template input is: 0.1pg to 100ng total RNA; 10fg to 100ng poly A(+) RNA. First-strand synthesis can be performed between 40°C and 52°C. Incubation at 50°C for 10 minutes usually yields the best results.

[0025] A. RNA template

[0026] The template RNA can be any ribonucleic acid of interest known or unknown to the practitioner. The template RNA can be artificially synthesized or isolated from a natural source. In some embodiments, the RNA template can be a ribonucleic acid, such as RNA, mRNA, piRNA, tRNA, rRNA, ncRNA, gRNA, shRNA, siRNA, snRNA, miRNA and snoRNA. Preferably, the RNA is mRNA. More preferably, the RNA is biologically active, or encodes a polypeptide with biological activity. The RNA template can also be present in any useful amount. In some embodiments, the RNA template concentration is 50pg / μL or less. It will be appreciated by those skilled in the art that other RNA template concentrations can be used in the present invention.

[0027] B. Reverse transcriptase

[0028] Reverse transcriptases useful in the present invention can be any polymerase that exhibits reverse transcriptase activity. Preferred enzymes include those that exhibit reduced RNase H activity. Several reverse transcriptases are known in the art and are commercially available (e.g., from Boehringer Mannheim Corp., Indianapolis, Indiana; Life Technologies, Inc., Rockville, Maryland; New England Biolabs, Inc., Beverly, Massachusetts; Perkin Elmer Corp., Norwalk, Connecticut; Pharmacia LKB Biotechnology, Inc., Piscataway, New Jersey; Qiagen, Inc., Valencia, California; Stratagene, La Jolla, California). In some embodiments, the reverse transcriptase can be avian myeloblastosis virus reverse transcriptase (AMV-RT), Moloney murine leukemia virus reverse transcriptase (M-MLV-RT), human immunovirus reverse transcriptase (HIV-RT), EIAV-RT, RAV2-RT, C. hydrogenoformans DNA polymerase, rTth DNA polymerase, SUPERSCRIPT I, SUPERSCRIPT II, ​​and mutants, variants, and derivatives thereof. It should be understood that a variety of reverse transcriptases can be used in the present invention, including reverse transcriptases not specifically disclosed above, without departing from the scope or preferred embodiments disclosed herein.

[0029] C. DNA polymerase

[0030] The DNA polymerase that can be used in the present invention can be any polymerase that can replicate a DNA molecule. Preferred DNA polymerases are thermostable polymerases that are particularly useful in PCR. Thermostable polymerases are isolated from various thermophilic bacteria, such as Thermus aquaticus (Taq), Thermus brockianus (Tbr), Thermus flavus (Tfl), Thermus ruber (Tru), Thermus thermophilus (Tth), Thermococcus litoralis (Tli) and other species of the Thermococcus genus, Thermoplasma acidophilum (Tac), Thermotoga neapolitana (Tne), Thermotoga maritime (Tma) and other species of the Thermotoga genus, Pyrococcus furiosus (Pfu), Pyrococcus wollastonii (Pyrococcus spp. woesei (Pwo) and other species of the Pyrococcus genus, Bacillus sterothemophilus (Bst), Sulfolobusacidocaldarius (Sac), Sulfolobus solfataricus (Sso), Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), and Methanobacterium thermoautotrophicum (Mth), and mutants, variants, or derivatives thereof.

[0031] Many DNA polymerases are known in the art and are commercially available (e.g., from Boehringer Mannheim Corp., Indianapolis, IN; Life Technologies, Inc., Rockville, MD; New England Biolabs, Inc., Beverly, MA; Perkin Elmer Corp., Norwalk, CT; Pharmacia LKB Biotechnology, Inc., Piscataway, NJ; Qiagen, Inc., Valencia, CA; Stratagene, La Jolla, CA). In some embodiments, the DNA polymerase can be Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT TM 、DEEPVENT TM And active mutants, variants or derivatives thereof. It should be understood that various DNA polymerases can be used in the present invention, including DNA polymerases not specifically disclosed above, without departing from the scope of the present invention or its preferred embodiments.

[0032] The reverse transcriptase can be present with the DNA polymerase in any suitable ratio. In some embodiments, the ratio of the reverse transcriptase per unit activity to the DNA polymerase is greater than or equal to 3. It will be appreciated by those skilled in the art that other ratios of reverse transcriptase to DNA polymerase can be used in the present invention.

[0033] D. Additives for improving reverse transcription efficiency

[0034] In some aspects, additives are provided to improve reverse transcription efficiency, improve inhibitor tolerance and / or prevent RNA degradation. Therefore, additives can be used for reverse transcription and PCR amplification reactions, such as qPCR. The reaction mixture containing additives may include other components, such as polymerase and / or reverse transcriptase. Including additives in the reaction mixture is sufficient to produce RT-qPCR results with an efficiency close to 100%, and at least 10%, or 40%, or 50%, or 100% higher than the control mixture lacking additives. These additives, such as poloxamer 188, histidine (e.g., histidine hydrochloride monohydrate), glutamic acid and / or its salts, reduce the inhibition of amplification reactions caused by heparin, formalin and / or tannic acid. In some aspects of the invention, additives, such as PVSA or its salts, can improve the efficiency of amplification reactions in the presence or absence of heparin, formalin and / or tannic acid.

[0035] In some embodiments, the additive is histidine or histidine hydrochloride monohydrate and can be included in the amplification reaction to improve RT efficiency, particularly in the presence of formalin and / or heparin. In the methods of the present invention, millimolar concentrations of histidine and / or histidine hydrochloride monohydrate have been found to be effective in improving the reaction efficiency of the amplification reaction. Histidine or histidine hydrochloride monohydrate at a concentration of about 0.01 mM to about 100 mM is used in the methods and kits of the present invention. In further embodiments, histidine or histidine hydrochloride monohydrate at a concentration of about 1 mM to about 50 mM, about 5 mM to about 40 mM, about 2.5 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, or about 15 mM to about 20 mM is used in the methods and kits of the present invention. Figure 3 As shown, the addition of histidine to the reaction mixture containing formalin improved the efficiency of the amplification reaction compared to a control lacking histidine.

[0036] In some embodiments, the additive is a poloxamer, such as poloxamer 188, which is included in the amplification reaction mixture to improve efficiency, especially in the presence of tannic acid. In the methods of the present invention, the poloxamer is added in an amount of about 0.01% to about 5% (v / v) of the reaction mixture. In various embodiments, the poloxamer is added in an amount of about 0.05% to about 4%, about 0.1% to about 3%, about 0.25% to about 2%, about 0.3% to about 1%, about 0.3% to about 0.75%, or about 0.3% to about 0.6%, or about 0.5% (v / v). Figure 4 As shown, the addition of poloxamer to the reaction mixture containing tannic acid improves the efficiency of the amplification reaction compared to a control lacking poloxamer. The concentration / amount of poloxamer present in the reaction mixture is provided as poloxamer content v / v %.

[0037] In further embodiments, glutamate or a salt thereof (e.g., an ammonium salt, a calcium salt, a magnesium salt, a monosodium salt, and / or a potassium salt) can be included in the amplification reaction to improve efficiency, particularly in the presence of heparin. For example, potassium glutamate is present in the amplification reaction mixture at a concentration of about 5 mM to about 250 mM, about 10 mM to about 400 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 15 mM to about 400 mM, about 15 mM to about 300 mM, about 15 mM to about 250 mM, about 15 mM to about 200 mM, or about 15 mM to about 150 mM.

[0038] In other embodiments, PVSA is included in the amplification reaction to improve RT efficiency and / or sensitivity. PVSA can be provided with a concentration (v / v) of about 0.0001% to about 0.002%, about 0.0001% to about 0.005%, about 0.0001% to about 0.004%, about 0.0001% to about 0.003%, about 0.0001% to about 0.002% or about 0.0001% to about 0.001%. The concentration / amount of PVSA present in the reaction mixture is provided with PVSA content v / v%.

[0039] In order to enhance RT, another additive that can be provided to the reaction mixture is one or more oligonucleotide primers as described herein. Disclosed one or more oligonucleotides can be used in combination with other disclosed additives or used alone. These oligonucleotide primers are five (5) to twelve (12) nucleotides in length and contain random nucleotides selected from adenine (A), cytosine (C), guanine (G) and thymine (T). Therefore, the oligonucleotide primers contain: X1-X2-X3-X4-X5, X1-X2-X3-X4-X5-X6, X1-X2-X3-X4-X5-X6-X7, X1-X2-X3-X4-X5-X6-X7-X8, X1-X2-X3-X4-X5-X6-X7-X8-X9, X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 (SEQ ID NO:21)、X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:22) or X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12(SEQ ID NO:23). If desired, the primer can contain one or more thiophosphate bonds at any position in the oligonucleotide. For example, one or more thiophosphate bonds can be introduced between the last, second to last and / or third to last nucleotide at the 5' or 3' end of the oligonucleotide. Alternatively, one or more thiophosphate bonds can be introduced between the last, second to last and / or third to last nucleotide at the 5' and 3' ends of the oligonucleotide. In some embodiments, the oligonucleotide primer is random. The oligonucleotide primers provided in other embodiments have high GC or high AT content. For the purpose of this application, at least 60% of the nucleotides in the high GC content primer are G and / or C. Similarly, at least 60% of the nucleotides in the high AT content primer are A and / or T.

[0040] Certain embodiments provide:

[0041] a) an X1-X2-X3-X4-X5 oligonucleotide sequence which is: a) randomly arranged A and T nucleotides; b) randomly arranged G and C nucleotides; c) at least one but less than five nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or d) at least one but less than five nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0042] b) an X1-X2-X3-X4-X5-X6 oligonucleotide sequence which is: a) randomly arranged A and T nucleotides; b) randomly arranged G and C nucleotides; c) at least one but less than six nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or d) at least one but less than six nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0043] c) an X1-X2-X3-X4-X5-X6-X7 oligonucleotide sequence which is: a) randomly arranged A and T nucleotides; b) randomly arranged G and C nucleotides; c) at least one but less than seven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or d) at least one but less than seven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0044] d) an X1-X2-X3-X4-X5-X6-X7-X8 oligonucleotide sequence which is: a) randomly arranged A and T nucleotides; b) randomly arranged G and C nucleotides; c) at least one but less than eight nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or d) at least one but less than eight nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0045] e) an X1-X2-X3-X4-X5-X6-X7-X8-X9 oligonucleotide sequence which is: a) randomly arranged A and T nucleotides; b) randomly arranged G and C nucleotides; c) at least one but less than nine nucleotides are randomly selected from the group consisting of: A, G, T and C, while the remaining nucleotides are randomly selected G and C nucleotides; or d) at least one but less than nine nucleotides are randomly selected from the group consisting of: A, G, T and C, while the remaining nucleotides are randomly selected A and T nucleotides;

[0046] f)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 (SEQ ID NO:21) an oligonucleotide sequence which is: a) randomly arranged A and T nucleotides; b) randomly arranged G and C nucleotides; c) at least one but less than ten nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or d) at least one but less than ten nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0047] g)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:22) an oligonucleotide sequence which is: a) randomly arranged A and T nucleotides; b) randomly arranged G and C nucleotides; c) at least one but less than eleven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or d) at least one but less than eleven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0048] h)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12(SEQ ID NO:23) an oligonucleotide sequence which is: a) randomly arranged A and T nucleotides; b) randomly arranged G and C nucleotides; c) at least one but less than twelve nucleotides are randomly selected from the group consisting of A, G, T and C, while the remaining nucleotides are randomly selected G and C nucleotides; or d) at least one but less than twelve nucleotides are randomly selected from the group consisting of A, G, T and C, while the remaining nucleotides are randomly selected A and T nucleotides.

[0049] In certain embodiments, the oligonucleotide sequence is dW n , wherein W is A or T and n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12. Other embodiments provide oligonucleotide sequence dS n , wherein S is G or C and n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12. Other embodiments provide oligonucleotide sequence dA n , dG n , dC n or dT n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12. Other embodiments provide a random oligonucleotide sequence X n (called RX n ), wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12 and X is any nucleotide disclosed in Section H, such as dNTP, dA, dC, dG or dT. Various combinations of any disclosed oligonucleotides can be added to the reaction mixture to enhance RT. In addition, the concentration of the primer added to the reaction mixture can be between about 1 μM to about 1000 μM, about 2.5 μM to about 750 μM, 5 μM to about 500 μM, 5 μM to about 250 μM, about 5 μM to about 100 μM, 10 μM to about 500 μM, 10 μM to about 250 μM, 10 μM to about 100 μM or 10 μM to about 50 μM.

[0050] Regarding the use of one or more oligonucleotides for enhancing reverse transcriptase activity, the method comprises adding an RNA template to a reaction mixture comprising one or more oligonucleotides disclosed herein, and incubating the mixture to synthesize all or part of a complementary DNA to the template RNA. Any combination of oligonucleotides can be used in this method. For example, the reaction mixture may comprise RX n and dW n ; RX n and dS n RX n and dW n RX n ,dS n and dW n RX n and dTn RX n ,dS n and dT n or RX n , dT n , dW n and dS n , wherein each n for a given oligonucleotide is independently selected from an integer selected from 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, n is independently selected from 5, 6, 7, 8, or 9. In other embodiments, n is independently selected from 6 or 7.

[0051] In addition to the reaction mixtures containing the additives at the respective concentrations described above, the present invention also provides stock solutions containing all or some of the above additives, wherein all components in the stock solution are 2X, 3X, 5X, 10X, 20X, 25X, 50X, 100X or other multiples of the above concentrations, so that a small amount of the stock solution can be added as part of the total volume of the final reaction mixture. For example, in some embodiments, the stock solution of the additives described herein can include a polymerase, nucleotides (e.g., dNTPs) and / or a buffer.

[0052] Certain embodiments of the invention provide an amplification reaction mixture comprising heparin, formalin, tannic acid, or any combination thereof.Alternative embodiments provide an amplification reaction mixture in which heparin, formalin, and tannic acid are absent.

[0053] E. Amplification reactions using the compositions of the present invention

[0054] As discussed herein, the present invention relates to amplification reactions, including but not limited to polymerase chain reaction (PCR), DNA ligase chain reaction (LCR), QBeta RNA replicase and amplification reactions based on RNA transcription (e.g., TAS and 3SR) and other amplification reactions as known to those skilled in the art. The polymerase chain reactions that can be performed using the compositions described herein include but are not limited to reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR) in a reaction mixture.

[0055] When the compositions of the present invention are components of an amplification reaction, the amplification reaction (e.g., polymerase chain reaction (PCR) method) exhibits improved efficiency and specificity. Typically, "efficiency" as discussed herein is represented by the amount of product produced under given reaction conditions. For example, in an efficient real-time PCR reaction, the PCR product should double in each cycle. As is known in the art, the efficiency of different amplification reactions can be calculated using different methods. For example, the exponential amplification of PCR is typically calculated using the equation X. n =X o *(1+E x )n (I) Determine, where X n is the number of target molecules at the nth cycle, X o is the initial number of target molecules, E x is the efficiency of target amplification and n is the number of cycles. Improvements in efficiency and specificity due to certain aspects of the present invention can be identified and quantified using assays known in the art.

[0056] F. Reaction mixture

[0057] The present invention also provides a reaction mixture comprising one or more additives of the present invention and at least one of the following: a reverse transcriptase; a polymerase; one or more dNTPs; a biological sample comprising a target nucleic acid. As also discussed herein, the reaction mixture may comprise heparin, formalin, tannic acid, or various combinations thereof. Alternative embodiments provide a reaction mixture in which heparin, formalin, and tannic acid are absent.

[0058] G. Kit

[0059] In one aspect, the present invention provides a kit for performing a nucleic acid amplification reaction. In some embodiments, the kit includes a polymerase and optional dNTPs, and at least one buffer containing an additive as disclosed herein. The kit may also include one or more primers and instructions for performing a nucleic acid amplification reaction using the kit components.

[0060] In some aspects, the instructions included in the kit of the present invention will include a typical amplification protocol, which includes the following steps: 95-98°C for about 30 seconds to about 2 minutes for 40 cycles (95-98°C for about 1 to about 5 seconds, 60°C for about 1 to about 5 seconds, detection step) and about 60°C to about 95°C melting cycle. It should be understood that the above exemplary protocol can be changed using parameters known in the art to optimize the nucleic acid amplification reaction to optimize the efficiency and specificity of different target nucleic acids. For example, the amplification of longer target nucleic acids may require longer incubation times and / or higher temperatures for efficient and specific amplification.

[0061] H. Nucleotide bases

[0062] Nucleotide bases that can be used in the present invention can be any nucleotide that can be used for nucleic acid polymerization. Nucleotide can be natural, rare, modified, derived or artificial. Nucleotide can be unlabeled, or can be detectably labeled by methods known to those skilled in the art (e.g., using radioisotopes, vitamins, fluorescent moieties or chemiluminescent moieties, digoxin (dioxigenin)). Preferably, nucleotide is a deoxynucleoside triphosphate, dNTP (e.g., dATP, dCTP, dGTP, dTTP, dITP, dUTP, α-thio-dNIT, biotin-dUTP, fluorescein-dUTP, digoxin-dUTP, 7-deaza-dGTP). dNTP is also well known in the art and can be commercially available from suppliers (e.g., from Boehringer Ingelheim, Indianapolis, Indiana; New England Biolabs, Beverly, Massachusetts; Pharmacia LKB Biotech Co., Ltd., Piscataway, New Jersey).

[0063] The nucleotides of the present invention may be present in any concentration. In some embodiments, the nucleotides may be present in an amount of about 1 μM to about 1000 μM. In other embodiments, the nucleotides may be present in an amount of about 10 μM to about 750 μM. In other embodiments, the nucleotides may be present in an amount of about 100 μM to about 500 μM. It will be appreciated by those skilled in the art that other concentrations of nucleotides may be used in the present invention.

[0064] I. Buffers and Salts

[0065] Buffers and salts useful in the present invention provide stable pH and ionic conditions suitable for nucleic acid synthesis (e.g., suitable for reverse transcriptase and DNA polymerase activity). A variety of buffers and salt solutions and improved buffers useful in the present invention are known in the art, including reagents not specifically disclosed in the art. Preferred buffers include, but are not limited to, TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, CAPS. Preferred salt solutions include, but are not limited to, the following solutions: potassium acetate, potassium sulfate, potassium chloride, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, and lithium acetate.

[0066] The buffer of the present invention can be present in any concentration. In some embodiments, the buffer is present in an amount of about 0.1 mM to about 1000 mM. In other embodiments, the buffer is present in an amount of about 1 mM to about 500 mM. In other embodiments, the buffer is present in an amount of about 5 mM to about 250 mM. It will be appreciated by those skilled in the art that other concentrations of buffer can be used in the present invention.

[0067] Various non-limiting embodiments include:

[0068] 1. A reaction mixture comprising a reverse transcriptase and one or more additives selected from glutamic acid or a salt thereof, poloxamer 188, histidine or a salt thereof and polyvinylsulfonic acid (PVSA), and one or more oligonucleotides selected from the following: X1-X2-X3-X4-X5, X1-X2-X3-X4-X5-X6, X1-X2-X3-X4-X5-X6-X7, X1-X2-X3-X4-X5-X6-X7-X8, X1-X2-X3-X4-X5-X6-X7-X8-X9, X1-X2-X3-X4-X5-X6-X7-X8-X9-X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 (SEQ ID NO:21)、X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:22) or X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 (SEQ ID NO:23);

[0069] 2. The reaction mixture as described in embodiment 1, wherein the additive is: a) glutamic acid or a salt thereof; b) histidine or a salt thereof; c) poloxamer 188; d) PVSA; e) glutamic acid or a salt thereof and histidine or a salt thereof; f) glutamic acid or a salt thereof and poloxamer 188; g) glutamic acid or a salt thereof and PVSA; h) histidine or a salt thereof and poloxamer 188; i) histidine or a salt thereof and PVSA; j) poloxamer 188 and PVSA; k) glutamic acid or a salt thereof, histidine or a salt thereof, and poloxamer 188; l) glutamic acid or a salt thereof, histidine or a salt thereof, and PVSA; m) histidine or a salt thereof, poloxamer 188 and PVSA; n) glutamic acid or a salt thereof, histidine or a salt thereof, poloxamer 188 and PVSA; or o) glutamic acid or a salt thereof, poloxamer 188 and PVSA;

[0070] 3. The reaction mixture of embodiment 1 or 2, wherein the glutamate salt is potassium glutamate and the histidine salt is histidine HCl monohydrate.

[0071] 4. The reaction mixture of any one of embodiments 1-3, wherein:

[0072] a) the poloxamer is present in an amount from about 0.01% to about 5% (v / v %);

[0073] b) histidine or a salt thereof is present at a concentration of about 1 mM to about 50 mM, about 5 mM to about 40 mM, about 2.5 mM to about 30 mM, about 5 mM to about 30 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, or about 15 mM to about 20 mM;

[0074] c) glutamate or a salt thereof is present at a concentration of about 5 mM to about 250 mM, about 10 mM to about 400 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 15 mM to about 400 mM, about 15 mM to about 300 mM, about 15 mM to about 250 mM, about 15 mM to about 200 mM, or about 15 mM to about 150 mM; and / or

[0075] d) PVSA is present in an amount (v / v %) of about 0.0001% to about 0.002%, about 0.0001% to about 0.005%, about 0.0001% to about 0.004%, about 0.0001% to about 0.003%, about 0.0001% to about 0.002%, or about 0.0001% to about 0.001%.

[0076] 5. The reaction mixture of embodiment 1, wherein the additive is one or more oligonucleotides.

[0077] 6. The reaction mixture of embodiment 5, wherein the one or more oligonucleotides contain one or more phosphorothioate bonds at any position within the one or more oligonucleotides.

[0078] 7. The reaction mixture of embodiment 6, wherein the one or more oligonucleotides contain one or more phosphorothioate bonds between the last, second to last and / or third to last nucleotides at the 5' end, the 3' end, or both the 5' and 3' ends of the oligonucleotides.

[0079] 8. The reaction mixture of embodiments 1, 5, 6 or 7, wherein the one or more oligonucleotides are a random sequence of nucleotides selected from A, G, T and C.

[0080] 9. The reaction mixture of embodiments 1, 5, 6, 7 or 8, wherein the one or more oligonucleotides are:

[0081] a) an X1-X2-X3-X4-X5 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than five nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than five nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0082] b) an X1-X2-X3-X4-X5-X6 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than six nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than six nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0083] c) an X1-X2-X3-X4-X5-X6-X7 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than seven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than seven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0084] d) an X1-X2-X3-X4-X5-X6-X7-X8 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than eight nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than eight nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0085] e) an X1-X2-X3-X4-X5-X6-X7-X8-X9 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than nine nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than nine nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0086] f)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10(SEQ ID NO:21) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than ten nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than ten nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0087] g)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:22) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than eleven nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than eleven nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; or

[0088] h)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 (SEQ ID NO:23) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than twelve nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than twelve nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides.

[0089] 10. The reaction mixture of embodiments 1, 5, 6, 7, 8 or 9, wherein the oligonucleotide has a high GC content and at least 60% of the nucleotides are G and / or C, or the oligonucleotide has a high AT content and at least 60% of the nucleotides are A and / or T.

[0090] 11. The reaction mixture of embodiment 2, 3 or 4, wherein the reaction mixture further comprises one or more oligonucleotides as additives.

[0091] 12. The reaction mixture of embodiment 11, wherein the one or more oligonucleotides contain one or more phosphorothioate bonds at any position within the oligonucleotides.

[0092] 13. The reaction mixture of embodiment 12, wherein the one or more oligonucleotides contain one or more phosphorothioate bonds between the last, second to last and / or third to last nucleotides at the 5' end, the 3' end, or both the 5' and 3' ends of the oligonucleotides.

[0093] 14. The reaction mixture of embodiments 11, 12 or 13, wherein the one or more oligonucleotides are a random sequence of nucleotides selected from A, G, T and C.

[0094] 15. The reaction mixture of embodiment 11, 12 or 13, wherein the one or more oligonucleotides are:

[0095] a) an X1-X2-X3-X4-X5 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than five nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than five nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0096] b) an X1-X2-X3-X4-X5-X6 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than six nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than six nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0097] c) an X1-X2-X3-X4-X5-X6-X7 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than seven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than seven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0098] d) an X1-X2-X3-X4-X5-X6-X7-X8 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than eight nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than eight nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0099] e) an X1-X2-X3-X4-X5-X6-X7-X8-X9 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than nine nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than nine nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0100] f)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 (SEQ ID NO:21) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than ten nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than ten nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides;

[0101] g)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:22) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than eleven nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than eleven nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; or

[0102] h)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 (SEQ ID NO:23) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than twelve nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than twelve nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides.

[0103] 16. A reaction mixture as described in embodiments 11-15, wherein one or more of the oligonucleotides have a high GC content and at least 60% of the nucleotides are G and / or C, or one or more of the oligonucleotides have a high AT content and at least 60% of the nucleotides are A and / or T.

[0104] 17. The reaction mixture of any one of embodiments 1 or 5-16, wherein the one or more oligonucleotides are selected from: dW n (wherein W is A or T and n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12), dS n (wherein S is G or C and n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12), dA n (wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12), dG n (wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12), dC n (wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12), dT n (wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12) or dW n ,dS n , dA n , dG n , dC n and dT n Any combination of .

[0105] 18. A reaction mixture as described in embodiment 17, wherein the concentration of any one or more of the oligonucleotides is 0.1 μM to about 1000 μM, about 2.5 μM to about 750 μM, about 5 μM to about 500 μM, about 5 μM to about 250 μM, about 5 μM to about 100 μM, about 10 μM to about 500 μM, about 10 μM to about 250 μM, about 10 μM to about 100 μM or about 10 μM to about 50 μM.

[0106] 19. The reaction mixture of any one of the preceding embodiments, wherein the reaction mixture comprises one or more of the following RT inhibitors: heparin, formalin and / or tannic acid.

[0107] 20. The reaction mixture of any one of embodiments 1-18, wherein the reaction mixture does not contain heparin, formalin and / or tannic acid.

[0108] 21. A method for reducing the inhibition of reverse transcriptase by formalin, wherein the method comprises: adding an RNA template to the reaction mixture described in any one of embodiments 1-19, incubating the mixture, and synthesizing complementary DNA of part or all of the template RNA, wherein the reaction mixture comprises formalin and at least histidine and / or its salt as an additive.

[0109] 22. A method for reducing the inhibition of reverse transcriptase by heparin, wherein the method comprises: adding an RNA template to the reaction mixture described in any one of embodiments 1-19, incubating the mixture, and synthesizing complementary DNA of part or all of the template RNA, wherein the reaction mixture comprises heparin and at least histidine and / or its salt and / or glutamic acid and / or its salt as additives.

[0110] 23. A method for reducing the inhibition of reverse transcriptase by tannic acid, wherein the method comprises: adding an RNA template to the reaction mixture described in any one of embodiments 1-19, incubating the mixture, and synthesizing complementary DNA of part or all of the template RNA, wherein the reaction mixture comprises tannic acid and at least poloxamer 188 as additives.

[0111] 24. A method for improving the efficiency and sensitivity of reverse transcriptase, wherein the method comprises: adding an RNA template to the reaction mixture described in any one of embodiments 1-19, incubating the mixture to synthesize complementary DNA of all or part of the template RNA, and the reaction mixture comprises PVSA as an additive.

[0112] 25. A method for enhancing reverse transcriptase activity, wherein the method comprises: adding an RNA template to a reaction mixture, incubating the mixture to synthesize complementary DNA of all or part of the template RNA, and the reaction mixture comprises one or more oligonucleotides as defined in any one of embodiments 5-10 as additives.

[0113] 26. The method of embodiment 25, wherein the one or more oligonucleotides are a combination of oligonucleotides selected from:

[0114] a) RX n and dW n ;

[0115] b) RX n and dS n ;

[0116] c) RX n , dW n and dS n ;

[0117] d) RX nand dT n ;

[0118] e)RX n , dT n and dS n ;or

[0119] f)RX n , dT n , dW n and dS n ,

[0120] wherein each n of a given oligonucleotide is independently selected from an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12.

[0121] 27. The method of embodiment 26, wherein for each oligonucleotide, each n is independently selected from 5, 6, 7, 8, or 9. In other embodiments, n is independently selected from 6 or 7.

[0122] 28. A method as described in embodiment 27, wherein for each oligonucleotide, each n is independently selected from 6 or 7.

[0123] 29. The method of any one of embodiments 25-28, wherein the reaction further comprises an additive of any one of embodiments 1-4.

[0124] 30. The method of any one of embodiments 25-28, wherein the one or more oligonucleotides contain one or more phosphorothioate bonds at any position within the one or more oligonucleotides.

[0125] 31. The method of embodiment 30, wherein the one or more oligonucleotides contain one or more phosphorothioate bonds between the last, second to last and / or third to last nucleotides at the 5' end, the 3' end, or both the 5' and 3' ends of the oligonucleotide.

[0126] All patents, patent applications, provisional applications, and publications cited or referenced herein, including all figures and tables, are hereby incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.

[0127] The following are examples of the process of illustrating the implementation of the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0128] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art should understand various modifications or changes made therefrom, and they are included in the subject matter and interests of this application and the scope of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0129] Example 1. Exemplary RT-qPCR Mixture

[0130] RT reaction buffer is prepared so that RT reaction can be carried out after mixing with thermostable RT (e.g., MMLV or engineered MMLV) and RNA template (e.g., Hela RNA). In 20 μl RT reaction mixture, there are 1u RT enzyme, 10u Rnasin (RNase inhibitor), 1.5mM of each nucleotide (e.g., dATP, dCTP, dGTP, dTTP), random hexamer, oligo-dT or gene-specific oligonucleotides of appropriate concentration (0.5 to 1uM), 4% glycerol, 0.01% detergent (e.g., Tween 20), with or without RT inhibitor (e.g., heparin, ethanol, guanidine hydrochloride, formalin, tannic acid, etc.) or additives (e.g., PVSA) that improve RT reaction efficiency or sensitivity. RT reaction is completed in T100 Thermo Cycler (Bio-Rad Laboratories Inc., California, Hercules). The RT reaction protocol was as follows: 50°C for 20 min, 95°C for 1 min, and incubation at 4°C.

[0131] After the RT reaction, the RT product was used for qPCR reaction using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). The input amount of RT product in the qPCR reaction ranged from 10% to 0.1% of the qPCR reaction, depending on the expression abundance of the target gene. 250 nM of the target gene primers were used (Table 3). The following protocol for qPCR was used on a CFC348 Thermo Cycler (Bio-Rad Laboratories, Hercules, CA): 95°C denaturation for 2 minutes, 40 cycles of 95°C denaturation for 5S, 60°C annealing / extension / reading for 30S. After the qPCR reaction, the Cq values ​​of the RT products with and without inhibitors were compared. When inhibitors were present, the Cq values ​​were generally delayed (larger values). The larger the Cq value relative to the RT product without inhibitor, the greater the inhibition of the RT reaction was observed.

[0132] Example 2. Effect of K-Glu on Heparin Tolerance

[0133] When 0.003u / μl inhibitor heparin (Sigma, H3149) was added to the RT reaction, the qPCR of the RT product was greatly delayed compared to the RT product without heparin. Both GAPDH and 18S showed similar results. When 100mM potassium glutamate (Sigma, C1149) was added to the RT reaction containing heparin, the qPCR results of the RT product did not show much delay compared to the RT product without heparin. This suggests that potassium glutamate in the RT reaction improves its tolerance to heparin. Other salts (e.g., (NH4)2SO4) can also improve heparin tolerance, but they may impair tolerance to other inhibitors (e.g., ethanol, guanidine hydrochloride, Calbiochem, 369075).

[0134] Example 3. Effect of Poloxamers on Tannic Acid Tolerance

[0135] When 0.1 μg / μl inhibitor tannic acid (Sigma, 403040) was added to the RT reaction, the qPCR results of the RT product were greatly delayed compared to the RT product without tannic acid. The human GAPDH target gene was detected in the qPCR reaction. When 0.5% poloxamer (Sigma, P5556) was added to the RT reaction containing the same concentration of tannic acid, the qPCR results of the RT product did not show much delay compared to the RT product without tannic acid. This indicates that poloxamer effectively improves the RT tolerance to tannic acid.

[0136] Example 4. Effect of His-HCl on formalin tolerance

[0137] Compared with the RT product without formalin, when 0.125% formalin (EMD, R04586-76) was added to the RT reaction, the qPCR results of the RT product were significantly delayed. The human GAPDH target gene was detected in the qPCR reaction. Compared with the RT product without formalin, when 15 to 20mM His-HCl (Sigma, H8125) was added to the RT reaction containing the same concentration of formalin, the qPCR results of the RT product did not show a significant delay. This shows that His-HCl effectively improves the RT tolerance to formalin.

[0138] Example 5. Effect of PVSA (polyethylene sulfonic acid) on RT yield and sensitivity

[0139] Compared with the RT product containing 0.0003% PVSA (Sigma, 278424), the qPCR results of this RT product were significantly delayed when RNA input in the RT reaction was limited (low input RNA template) and PVSA was not included. Human GAPDH target gene was detected in the qPCR reaction.

[0140] Example 6. Enhancement of reverse transcription of high and low GC targets

[0141] Compared to the RT products without adding low / high GC for low or high GC targets (target genes in Table 3), the qPCR results of the RT products in the RT reactions with added low / high GC random primers were significantly improved (earlier Cq, smaller Cq value).

[0142] Table 1. Enhancement of reverse transcription of high and low GC targets. Reverse transcription was performed using random hexamers only or with the addition of different low / high GC random primers. cDNA was quantified by qPCR. dCq was calculated between the conditions using random primers only and adding low / high GC random primers in reverse transcription; dCq(-Cq(RH)-Cq(test condition). Negative dCq values ​​indicate that the Cq of the test condition was earlier and reverse transcription was enhanced. RH: DNA random hexamer; dW7; oligo dW septamer; dS6; oligo dS hexamer. dT6: oligo dT hexamer. The concentrations of oligonucleotides used are indicated. *: -0.5 ≥ dCq ≥ -0.9; **: dCq ≤ -0.91.

[0143]

[0144] Table 2. GC content of different target RNAs

[0145] GC content (%) NGFRAP198 40 SNRPE109 35.8 TXN192 29 RHPN2 27 SBNO2 GC3 74.8 CBP hgc1 78.5 CBP hgc2 81.7 CBP hgc3 78.3

[0146] Table 3. Primer sequences used in the study of this application

[0147]

[0148]

Claims

1. A reaction mixture comprising: one or more additives selected from the group consisting of glutamic acid or its salt, poloxamer 188, histidine or its salt and polyvinyl sulfonic acid, and one or more oligonucleotides, wherein the concentration of glutamic acid or its salt is 5 mM to 250 mM, The content of poloxamer 188 v / v% is 0.01% to 5%, wherein the concentration of histidine or its salt is 1 mM to 50 mM, The content of polyvinyl sulfonic acid v / v% is 0.0001% to 0.002%, wherein the one or more oligonucleotides are selected from: X1-X2-X3-X4-X5, X1-X2-X3-X4-X5-X6, X1-X2-X3-X4-X5-X6-X7, X1-X2-X3-X4-X5-X6-X7-X8, X1-X2-X3-X4-X5-X6-X7-X8-X9, X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 (SEQ ID NO:21)、X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:22) or X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 (SEQ ID NO:23), in: a) an X1-X2-X3-X4-X5 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than five nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than five nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; b) an X1-X2-X3-X4-X5-X6 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than six nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than six nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; c) an X1-X2-X3-X4-X5-X6-X7 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than seven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than seven nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; d) an X1-X2-X3-X4-X5-X6-X7-X8 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than eight nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than eight nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; e) an X1-X2-X3-X4-X5-X6-X7-X8-X9 oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than nine nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than nine nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; f)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 (SEQ ID NO:21) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than ten nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than ten nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; g)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:22) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than eleven nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than eleven nucleotides are randomly selected from the group consisting of A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides; or h)X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 (SEQ ID NO:23) an oligonucleotide sequence which is: i) randomly arranged A and T nucleotides; ii) randomly arranged G and C nucleotides; iii) at least one but less than twelve nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected G and C nucleotides; or iv) at least one but less than twelve nucleotides are randomly selected from the group consisting of: A, G, T and C, and the remaining nucleotides are randomly selected A and T nucleotides.

2. The reaction mixture of claim 1, wherein The one or more additives are: Poloxamer 188 in an amount of 0.01% to 5% v / v%; Potassium glutamate at a concentration of 5 mM to 250 mM; and Polyethylene sulfonic acid in an amount v / v% ranging from 0.0001% to 0.002%.

3. The reaction mixture of claim 1, wherein the glutamate salt is potassium glutamate and the histidine salt is histidine HCl monohydrate.

4. The reaction mixture of claim 1, wherein The one or more additives are: Poloxamer 188 in an amount of 0.1% to 3% v / v%; Potassium glutamate at a concentration of 15 mM to 150 mM; and Polyethylene sulfonic acid in an amount v / v% of 0.0001% to 0.001%.

5. The reaction mixture of claim 1, wherein the one or more oligonucleotides contain one or more phosphorothioate bonds at any position within the one or more oligonucleotides.

6. The reaction mixture of claim 5, wherein the one or more oligonucleotides contain one or more phosphorothioate bonds between the last, second to last and / or third to last nucleotides at the 5' end, the 3' end, or both the 5' and 3' ends of the oligonucleotides.

7. The reaction mixture of claim 1 or 2, wherein the reaction mixture further comprises dNTPs.

8. The reaction mixture of claim 1, wherein the reaction mixture further comprises one or more oligonucleotides selected from the group consisting of: n , wherein W is A or T and n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dS n , wherein S is G or C and n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dA n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dG n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dC n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dT n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; or dW n ,dS n , dA n , dG n , dC n and dT n Any combination of .

9. The reaction mixture of claim 7, further comprising one or more oligonucleotides selected from the group consisting of: n , wherein W is A or T and n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dS n , wherein S is G or C and n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dA n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dG n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dC n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; dT n , wherein n is an integer selected from 5, 6, 7, 8, 9, 10, 11 or 12; or dW n ,dS n , dA n , dG n , dC n and dT n Any combination of .

10. The reaction mixture of claim 1, wherein said one or more of said oligonucleotides have a high GC content and at least 60% of the nucleotides are G and / or C, or said one or more of said oligonucleotides have a high AT content and at least 60% of the nucleotides are A and / or T.

11. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 0.1 μM to 1000 μM.

12. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 2.5 μM to 750 μM.

13. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 5 μM to 500 μM.

14. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 5 μM to 250 μM.

15. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 5 μM to 100 μM.

16. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 10 μM to 500 μM.

17. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 10 μM to 250 μM.

18. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 10 μM to 100 μM.

19. The reaction mixture of claim 1, wherein the concentration of any one or more oligonucleotides is 10 μM to 50 μM.

20. The reaction mixture of claim 1, wherein the reaction mixture comprises one or more of the following RT inhibitors: heparin, formalin, and / or tannic acid.

21. The reaction mixture of claim 1, wherein the reaction mixture does not contain heparin, formalin and / or tannic acid.

22. A method for reducing the inhibition of reverse transcriptase by formalin, wherein the method comprises adding an RNA template to a composition comprising reverse transcriptase and a reaction mixture as claimed in claim 1, hatching the mixture, and synthesizing a complementary DNA of part or all of the template RNA, wherein the reaction mixture comprises formalin and at least histidine and / or its salt as an additive.

23. A method for reducing heparin inhibition of reverse transcriptase, wherein the method comprises adding an RNA template to a composition comprising reverse transcriptase and a reaction mixture as claimed in claim 1, incubating the mixture, and synthesizing complementary DNA to part or all of the template RNA, wherein the reaction mixture comprises heparin and at least histidine and / or a salt thereof and / or glutamic acid and / or a salt thereof as additives.

24. A method for reducing the inhibition of reverse transcriptase by tannic acid, wherein the method comprises adding an RNA template to a composition comprising reverse transcriptase and a reaction mixture as claimed in claim 1, incubating the mixture, and synthesizing complementary DNA to part or all of the template RNA, wherein the reaction mixture comprises tannic acid and at least poloxamer 188 as additives.

25. A method for improving the efficiency and sensitivity of a reverse transcriptase, wherein the method comprises adding an RNA template to a composition comprising a reverse transcriptase and a reaction mixture as claimed in claim 1, incubating the mixture to synthesize complementary DNA to all or part of the template RNA, wherein the reaction mixture comprises polyvinyl sulfonic acid as an additive.

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