WO2017143873A1 - Isothermal nucleic acid amplification method - Google Patents

Abstract

Disclosed is an isothermal nucleic acid amplification method, carried out under a constant temperature, wherein a forward primer and a target are annealed and are then extended under the effect of a polymerase to generate an amplification target; meanwhile, a reverse primer and the amplification target are annealed by utilizing the principle of dynamic dissociation between two strands, and are then extended under the effect of the polymerase to generate a double-stranded amplification product. The generated double-stranded amplification product can be reused as a substrate for another circulation to implement cyclic amplification, thereby achieving exponential amplification.

Description

Method for isothermal nucleic acid amplification Background technique

In recent years, nucleic acid amplification methods have received extensive attention. Currently, Polymerase Chain Reaction (PCR) is the most commonly used method for amplifying nucleic acid sequences in vitro. The PCR method uses a small amount of double-stranded nucleic acid as a template to achieve exponential amplification, and the amplification result has the advantage of high sensitivity, so the method is widely used as a tool for cloning or nucleic acid amplification. However, the PCR method clearly has the following problems: each cycle of PCR includes three steps of denaturation, annealing and extension. Therefore, it is necessary to use an instrument with precise temperature regulation, which is difficult to apply at the bedside or outside of the hospital; Refolding temperature to improve specificity, work is more complicated and complicated; reaction takes a long time; thus it can not meet the simple and fast requirements in the field of real-time detection.

In order to overcome the shortcomings of the PCR method, many laboratories have tried to develop a series of isothermal nucleic acid amplification methods without thermal denaturation since the early 1990s, such as Strand Displacement Amplification (SDA) and ring-mediated isothermal expansion. (Loop Mediated Isothermal Amplification, LAMP), Cross Priming Amplification (CPA), etc. The common feature of these methods is that the amplification reaction is simple and rapid at a specific temperature, which greatly reduces the instrument. Requirements.

Strand Displacement Amplification (SDA) is a method developed by Walker et al. in 1992 to detect double-stranded nucleic acid amplification. This method is mainly based on the interaction of a restriction enzyme and a DNA polymerase. The principle is to first convert a double-stranded DNA into a single strand using a thermal denaturation method, and a primer containing a restriction endonuclease recognition sequence binds to a single-stranded template and is extended by a DNA polymerase. Forming a complementary strand of the template, the addition of the outer primer strand replaces the template complementary strand formed, and another primer with a restriction enzyme recognition sequence binds to and extends the complementary strand of the formed template, and a specific base of thio is used in the experiment. The endonuclease cleaves a strand in the double strand to create a nick, the polymerase synthesizes a new strand at the nick and replaces the original strand while filling in the cleavage site, and the replaced strand is complementary to the amplification primer. Cycle amplification. Chain displacement amplification is highly sensitive and can be rapidly amplified to obtain single-stranded DNA, but this method requires an initial thermal denaturation step; the reaction also requires restriction enzymes, which adds additional cost and requires the participation of four primers. It is easy to cause non-specific reactions; the limitations of detection methods limit its application range.

Loop Mediated Isothermal Amplification (LAMP) is a new method for in vitro amplification of nucleic acid-specific sequences established by Japanese scholar Notomi et al. in 2000. The method mainly uses four specific primers. The six specific regions of the target DNA are identified, and rapid isothermal amplification of the nucleic acid is achieved by two loop structures and a strand displacement reaction. The LAMP reaction process includes a dumbbell template synthesis stage, a cycle amplification stage, an elongation and a recycle stage. The method is rapid in response and has good sensitivity and specificity. However, the design of the four primers is more difficult, and requires special design software, which requires high requirements for designers, which may cause aerosol contamination and lead to false positive results, and is used for qualitative rather than quantitative detection in testing. The above shortcomings have limited the promotion and application of the technology to some extent.

Cross Priming Amplification (CPA) is a method of constantly amplifying target nucleic acid sequences. The method mainly designs 5 specific primers for 5 regions of the target gene, cross-interleaving sequences at the tail ends of the primers, and DNA aggregation with strand displacement activity. The enzyme is subjected to extension and displacement, and is amplified by linear structure or secondary structure, and multiple primer hybridization sites are generated by repeated hybridization, extension and self-hybrid folding and extension of the amplification product, so that the same template can be used. A plurality of amplification reactions are simultaneously performed to complete the nucleic acid amplification reaction under constant temperature conditions, and the method has the advantages of not requiring thermal denaturation of the template, multiple temperature cycling processes, rapid reaction, and the like. However, the method has a large number of primers, and is easy to produce a non-specific reaction, and the obtained amplification product is complicated, and can only be qualitatively detected by agarose gel electrophoresis and cannot be quantitatively analyzed.

Summary of the invention

The invention provides a method for isothermal nucleic acid amplification, which solves the problems that the prior art isothermal nucleic acid amplification method requires multiple primers, multiple enzymes, and poor amplification effect on small fragments.

The invention establishes a nucleic acid amplification reaction by designing two forward and reverse primers, utilizing the dynamic dissociation principle between the double strands and the action of the strand displacement DNA polymerase under the isothermal condition to complete the nucleic acid amplification reaction. A novel rapid and sensitive method for nucleic acid amplification.

According to the patent 201510501218.X, the Bst DNA polymerase has both reverse transcription and strand displacement activities, and the RNA can be reverse transcribed into cDNA under the action of the substrate, and the strand displacement amplification reaction is carried out by using Bst DNA polymerase. The whole system is completely expanded under isothermal conditions without additional reverse transcriptase, which integrates reverse transcription and amplification, reduces the amount of enzyme, and greatly shortens the reaction time and reduces the reaction. the cost of.

In order to achieve the above object, a method for isothermal nucleic acid amplification comprises the following steps:

(1) design two forward and reverse primers;

(2) the forward primer hybridizes with the amplified target nucleic acid sequence, and polymerizes and extends under the action of a polymerase to obtain an amplification product;

(3) The amplification product obtained in the step (2) is dynamically dissociated at the reaction temperature, so that the reverse primer hybridizes with the amplification product and extends to obtain an amplification product of the two primers;

(4) The amplification products of the two primers are continuously hybridized and extended at the reaction temperature, and finally a large amount of amplification products are obtained.

Further, the Tm value of the primer is ≥ the reaction temperature, and the primer length is between 15-30 nucleotides.

The reaction temperature is 55-73 ° C, and the preferred reaction temperature is 65 ° C.

Further, the amplified target nucleic acid sequence may be DNA or RNA, and the amplified target nucleotide sequence is ≤100 bp, preferably ≤60 bp.

Therefore, the shortest length of the amplified target nucleotide sequence of the present invention may be the sum of the lengths of amplification of the two primers, and the principle of dynamic dissociation between the double strands under the condition of high reaction temperature during the amplification process is utilized. .

The method of the present invention is carried out at a constant temperature. First, the forward primer is annealed to the target, and then extended by the polymerase to generate an amplification target, and the reverse primer and amplification are simultaneously utilized by the principle of dynamic dissociation between the two strands. The target is annealed and then extended by the polymerase to obtain a double-stranded amplification product, and the resulting double-stranded amplification product can be reused as a substrate for another cycle, and is continuously circulated and amplified, thereby causing exponential amplification. Therefore, the reaction The design of the primer Tm value is especially critical, that is, the designed primer has a Tm value higher than the reaction temperature to ensure that the primer can completely anneal to the target.

Further, in order to speed up the reaction, we also add substances that promote chain exchange, such as PEG-200, extremely high heat stable single-stranded binding protein (ET SSB), etc., to ensure rapid reaction.

Further, the polymerase of the present invention is a polymerase which is stable at the reaction temperature, preferably a polymerase having no exonuclease activity.

Preferably, when the target nucleotide sequence is RNA, the polymerase has reverse transcription activity or the enzyme reaction temperature of the reverse transcriptase is consistent with the reaction temperature of the polymerase.

The beneficial effects of the present invention are mainly embodied in the following aspects:

1) The method of the present invention can be completed under isothermal conditions, and isothermal means that the reaction temperature of each step in the whole process of the technical solution is constant, and each step is performed at a constant temperature without repeated lifting and lowering. The invention does not need to adjust the temperature up and down in the whole process of nucleic acid amplification, and can be implemented only by using a device capable of maintaining a constant temperature, and the operation is simple;

2) The method of the present invention requires only one polymerase in the amplification process, especially when the RNA is amplified, no additional reverse transcriptase is added, and the reverse transcription and amplification are integrated, and the enzyme is reduced. The amount of use, and greatly shorten the reaction time, while reducing the cost of the reaction;

3) The technical solution of the present invention requires only two forward and reverse primers in the amplification process, and does not need the four primers described in the prior art LAMP method, and the experimental design is simple and the input cost is low.

4) The method of the invention has good amplification effect on small fragments, and is more suitable for detecting mutated RNA viruses under isothermal conditions.

DRAWINGS

Figure 1 is a schematic diagram of the method of the present invention;

2 is a schematic diagram of the principle of the method of the present invention; wherein, FIG. 2-a is a fluorescence verification diagram, wherein A indicates that a target, an enzyme, and a primer are added to the reaction system; B indicates that only an enzyme and a primer are added; and C indicates that only a primer is added. Figure 2-b shows the PAGE electrophoresis verification chart.

Lane 450: DNA ladder;

Lane 1: only two primers containing 500 nM;

Lane 2: Two primers and DNA polymerase containing 500 nM

Lane 3: Two primers containing 1 nM HCV-DNA, 500 nM, also added to DNA polymerase

Figure 3 is a graph showing amplification results of target nucleotide sequences of different lengths, wherein

An amplification map showing the length of the amplified product of 45 bp, --- NTC indicates an amplification map of the template with a length of 45 bp instead of water.

An amplification map showing an amplification template length of 66 bp, and -b NTC indicates an amplification map of a template having a length of 66 bp instead of water.

An amplification map showing an amplification product of 87 bp in length, --c NTC indicates an amplification map of a template having a length of 87 bp instead of water.

Figure 4 is a graph showing the results of amplification of different amounts of PEG-200, wherein

Indicates an amplification map of 20% PEG-200 added to the system.

Represents an amplification map of 10% PEG-200 added to the system.

Indicates the addition of 5% PEG-200 to the system.

An amplification plot showing the addition of 0% PEG-200 to the system.

Figure 5 is a diagram showing the results of double-stranded DNA amplification, wherein

An amplification map showing a target concentration of 10 ^-8 M,

An amplification map showing a target concentration of 10 ^-9 M,

An amplification map showing a target concentration of 10 ^-10 M,

An amplification map showing a target concentration of 10 ^-11 M,

An amplification map showing a target concentration of 0M.

Figure 6 is a graph showing the results of amplification of RNA at different concentrations, wherein

An amplification map showing a target concentration of 10 ^-9 M,

An amplification map showing a target concentration of 10 ^-10 M,

An amplification map showing a target concentration of 10 ^-11 M,

Indicates an amplification map when the target concentration is 0M.

Figure 7 is a graph showing the results of amplification of vent DNA polymerase.

Figure 8 is a graph showing the results of anti-interference amplification, wherein

An amplification map showing the addition of 10% bovine serum to 16S rRNA with a target concentration of 10 ^-9 M,

An amplification map showing the addition of 10% bovine serum to 16S rRNA with a target concentration of 10 ^-10 M,

An amplification map showing the addition of 10% bovine serum to 16S rRNA with a target concentration of 10 ^-11 M,

Indicates an amplification map when the detection concentration is 0M.

detailed description

Example 1: The feasibility of the verification method and the correctness of its principle.

In this example, the DNA sequence of the synthesized HCV virus is used as a target nucleotide (sequence is 5'-GCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGT-3' (SEQ ID NO. 1), and two primers P1 (sequence 5'-ACCTCCCGGGGCACT-3' are utilized. (SEQ ID NO. 2)), P2 (sequence 5'-GCCTGATAGGGTGCTTGCG-3' (SEQ ID NO. 3)) was subjected to constant temperature amplification, and the feasibility and principle of the method were verified by fluorescence signal and electrophoresis results.

The 10 μL reaction system is as follows:

1 Primers P1 and P2 are 5×10 ^-7 M, respectively

2dNTPs (10mM): 0.5μL

31×ThermoPol Reaction Buffer Reaction Buffer:

(20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, pH 8.8 @ 25 ° C)

4Eva Green (20×): 0.25 μL

5Bst 2.0 WarmStart ^TM DNA polymerase (8U/μL): 0.1μL

Then added to a final concentration of 1 × 10 ^-9 M of HCV-DNA target using the BioRad CFX96 ^TM real-time PCR instrument to detect the fluorescence signal collected once every minute, reaction at 65 ℃, amplification results shown in Figure 2a.

After the above reaction for 1 h, the product was subjected to 17.5% PAGE electrophoresis, and a 5 μL voltage of 135 V was applied, and electrophoresis was performed for 55 min, and EB staining was performed. The results are shown in Fig. 2b.

The results showed that the method could only react under the action of target, polymerase and primers and obtain the corresponding amplification products, thus verifying the feasibility and correctness of the method.

Example 2: Amplification of target nucleotide sequences of different lengths

In this example, the method of the present invention is used to amplify 16S rDNA of Escherichia coli of different lengths, and three pairs of primers are designed, and these primers can amplify DNA product sequences of different lengths (45 bp, 66 bp, 87 bp).

The reaction system is:

1 primers P1 and P2 are 10 ^-6 M, respectively

2dNTPs (10mM): 0.5μL

31×ThermoPol Reaction Buffer Reaction Buffer:

(20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, pH 8.8 @ 25 ° C)

4Eva Green (20×): 0.25 μL

5Bst 2.0 WarmStart ^TM DNA polymerase (8U/μL): 0.1μL

Further, 1 μL of 1×10 ^-10 M 16S rDNA target was added, and 10 μL of the final reaction system was added. Bole CFX96 ^TM using real-time PCR instrument to detect the fluorescence signal collected once every minute, reaction at 65 ℃, amplification results shown in Figure 3.

1) wherein 16S rDNA of 45 bp in length (sequence 5'-CAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACAC-3' (SEQ ID NO. 4)),

Primer P1 (sequence is 5'-CAAGGCGACGATCCCTAGCT-3' (SEQ ID NO. 5)),

Primer P2 (sequence is 5'-GTGTGGCTGGTCATCCTCTCAGAC-3' (SEQ ID NO. 6));

2) 16S rDNA of a length of 66 bp (sequence 5'-GTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACAC-3' (SEQ ID NO. 7)),

Primer P1 (sequence is 5'-GTGTGGCTGGTCATCCTCTCAGAC-3' (SEQ ID NO. 8)),

Primer P2 (sequence is 5'-GTTGGTGGGGTAACGGCTCAC-3' (SEQ ID NO. 9));

3) Amplification of a 16 bp length 16S rDNA (sequence 5'-CAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGT-3' (SEQ ID NO. 10)),

Primer P1 (sequence is 5'-ACTGCTGCCTCCCGTA-3' (SEQ ID NO. 11)),

Primer P2 (sequence is 5'-CAAGGCGACGATCCCTAGCT-3' (SEQ ID NO. 12)).

The results showed that as the length of the amplified product increased, the amplification efficiency decreased gradually. It can be seen from the amplification results that the amplification effect of the 45 bp product in the three different length sequences is the best, indicating that the invention is small. The fragment amplification effect is good, and it is more suitable for detecting the susceptible RNA virus under isothermal conditions.

Example 3 Amplification of PEG-200 in different amounts:

Since PEG promotes the chain exchange reaction, in order to verify whether PEG promotes the present invention, in the present example, different ratios of PEG-200 are separately added for verification, and the target nucleotide to be amplified is PBS plasmid (sequence is 5'-ATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGC-3' (SEQ ID NO. 13)), the detection primers were P1 (sequence 5'-GCCGTCGTTTTACAACGTCGTGA-3' (SEQ ID NO. 14)) and primer P2 (sequence 5'-ATTAAGTTGGGTAACGGCAGGGT-3) '(SEQ ID NO. 15)). 10 μL reaction system;

1 Primers P1 and P2 are 1.0×10 ^-6 M, respectively.

2dNTPs (10mM): 0.5μL

31×ThermoPol Reaction Buffer Reaction Buffer:

(20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, pH 8.8 @ 25 ° C)

4Eva Green (20×): 0.25 μL

5Bst 2.0 WarmStart ^TM DNA polymerase (8U/μL): 0.1μL

61×10 ^-8 M PBS template

Then, 0%, 5%, 10%, and 20% PEG was added to the system, and water was added to make the reaction system 10 μL.

The detection was performed using a Bole CFX96 ^TM real-time PCR instrument, and the fluorescence signal was collected every minute, and the reaction was carried out at 65 ° C. The amplification results are shown in FIG. 5 .

The results showed that the addition of different proportions of PEG-200 had a certain effect on the reaction. It can be seen from the amplification results that the addition of 10% PEG-200 can significantly accelerate the reaction rate. Therefore, the present invention will 10% PEG. -200 acts as a promoter to speed up the reaction.

Example 4, Double-stranded nucleic acid amplification:

In this example, different concentrations of PBS plasmid were used as target nucleotides (sequence 5'-ATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGC-3' (SEQ ID NO. 16)). The two primers P1 (sequence 5'-GCCGTCGTTTTACAACGTCGTGA-3' (SEQ ID NO. 17)) and P2 (sequence 5'-ATTAAGTTGGGTAACGCCAGGGT-3' (SEQ ID NO. 18)) were subjected to constant temperature amplification.

10 μL reaction system;

1 Primers P1 and P2 are 1.0×10 ^-6 M, respectively.

2dNTPs (10mM): 0.5μL

31×ThermoPol Reaction Buffer Reaction Buffer:

(20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, pH 8.8 @ 25 ° C)

410% PEG-200

5Eva Green (20×): 0.25 μL

6Bst 2.0 WarmStart ^TM DNA polymerase (8U/μL): 0.1μL

Further, different concentrations of PBS target (1 × 10 ^-8 M, 1 × 10 ^-9 M, 1 × 10 ^-10 M, 1 × 10 ^-11 M and 0 M) were added, and the reaction system was 10 μL.

Bole CFX96 ^TM using real-time PCR instruments detect a fluorescent signal collected once every minute, reaction at 65 ℃, amplification results shown in Figure 5.

The results show that the present invention can perform better amplification of the PBS target having a helical double-strand structure, and thus the method can also be applied to the amplification of other double-stranded targets.

Example 5: Amplification of RNA at different concentrations:

In this experiment, Escherichia coli 16S rRNA was used as an amplification template (sequence is 5'-GCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCG-3' (SEQ ID NO. 19)), and two primers P1 (sequence 5'-CGCTCGTTGCGGGACTTAACC-3' (SEQ ID NO. 20)), P2 (sequence 5'-GCTGTCGTCAGCTCGTGTTG-3' (SEQ ID NO. 21)) was subjected to constant temperature amplification. 10 μL reaction system;

1 Primers P1 and P2 are 1.0×10 ^-6 M, respectively.

2dNTPs (10mM): 0.5μL

31×ThermoPol Reaction Buffer Reaction Buffer:

(20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, pH 8.8 @ 25 ° C)

410% PEG-200

5Eva Green (20×): 0.25 μL

6Bst 2.0 WarmStart ^TM DNA polymerase (8U/μL): 0.1μL

Further, 16S rRNA (1×10 ^-8 M, 1×10 ^-9 M, 1×10 ⁻¹⁰ M, 1×10 ^-11 M and 0 M) of different concentrations of Escherichia coli was added, and the reaction system was 10 μL.

Bole CFX96 ^TM using real-time PCR instruments detect a fluorescent signal collected once every minute, reaction at 65 ℃, amplification results shown in Figure 6.

The results showed that the present invention utilizes the characteristic that Bst DNA polymerase has reverse transcription activity, and the extracted Escherichia coli 16S rRNA is used as a target, and is directly reverse transcribed into cDNA for subsequent amplification reaction, that is, reversed under isothermal conditions. The integration of recording and amplification allows the present invention to achieve one-step amplification of RNA.

Example 6. Amplification of other polymerases

10 μL reaction system:

1 Primers P1 and P2 are 1.0×10 ^-6 M, respectively.

2dNTPs (10mM): 0.5μL

31×ThermoPol Reaction Buffer Reaction Buffer:

(20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, pH 8.8 @ 25 ° C)

4Eva Green (20×): 0.25 μL

5Vent _R (exo-) DNA polymerase (0.4U)

Further, 1 μL of 1×10 ^-7 M HCV-DNA target was added, and 10 μL of the final reaction system was added. The detection was performed using a Bole CFX96 ^TM real-time PCR instrument, and the fluorescence signal was collected every minute, and the reaction was carried out at 65 ° C. The amplification results are shown in FIG. 7 .

The results show that other enzymes which are stable at the reaction temperature can be applied to the present invention. The method of nucleic acid amplification.

Embodiment 6. Verification of the anti-interference ability of the present invention:

On the basis of Example 5, 10% bovine serum was added to the reaction system to verify the anti-interference ability of the method of the present invention, and the amplification results are shown in Fig. 8.

The results showed that the addition of 10% bovine serum did not inhibit the amplification reaction, and the amplification results showed good amplification curves as in the reaction buffer, indicating that the invention has good anti-interference in complex reaction environments. Ability, can be well applied in the detection of actual samples.

Claims (10)

1. A method of isothermal nucleic acid amplification comprising the steps of:

   (1) design two forward and reverse primers;

   (2) the forward primer hybridizes with the amplified target nucleic acid sequence, and polymerizes and extends under the action of a polymerase to obtain an amplification product;

   (3) The amplification product obtained in the step (2) is dynamically dissociated at the reaction temperature, so that the reverse primer hybridizes with the amplification product and extends to obtain an amplification product of the two primers;

   (4) Steps (2) and (3) are repeatedly cycled to finally obtain a large amount of amplification product.
2. A method of isothermal nucleic acid amplification according to claim 1, wherein said forward and reverse primers have a Tm value ≥ a reaction temperature, and a primer length of between 15 and 30 nucleotides.
3. A method of isothermal nucleic acid amplification according to claim 1 or 2, wherein the reaction temperature is constant throughout the process, and the reaction temperature ranges from 55 to 73 °C.
4. A method of isothermal nucleic acid amplification according to claim 3, wherein the reaction temperature is 65 °C.
5. A method of isothermal nucleic acid amplification according to claim 1, wherein the amplified target nucleic acid sequence can be a DNA sequence or an RNA sequence.
6. A method of isothermal nucleic acid amplification according to claim 1 or 5, wherein the target nucleotide sequence is ≤ 100 bp in length.
7. A method of isothermal nucleic acid amplification according to claim 1, wherein the method further comprises adding a substance which promotes a chain exchange reaction to the reaction system, and the substance for promoting the chain exchange reaction is PEG-200 or Very high heat stable single-stranded binding protein.
8. A method of isothermal nucleic acid amplification according to claim 1, wherein said polymerase is a polymerase which is stable at the reaction temperature.
9. A method of isothermal nucleic acid amplification according to claim 1 or 8, wherein the polymerase is a polymerase having no exonuclease activity.
10. A method for isothermal nucleic acid amplification according to claim 1 or 8, wherein when the target nucleotide sequence is RNA, the polymerase has a reverse transcription activity or an enzyme reaction temperature of reverse transcriptase The reaction temperature with the polymerase is the same.

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