( Powerful high-fidelity R...)prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification

Powerful high-fidelity RT-PCR systems in two convenient formats

Michael Borns Janice Cline Kris Connors Ronda Allen Holly Hogrefe
Stratagene

Stratagenes prostar RT-PCR systems are powerful systems that permit efficient, high-fidelity amplification of RNA in two convenient formats. The ProSTAR Ultra HF RT-PCR system features pfuturbo DNA polymerase * for amplifying cDNAs with the highest replication fidelity possible using a versatile two-tube format. The ProSTAR HF Single-Tube RT-PCR system combines the sensitivity and accuracy of the TaqPlus Precision DNA polymerase ** blend with the convenience of a one-step, one-tube procedure. These two systems are ideally suited for subcloning, expression, and sequencing of DNA coding regions and for reproducibly detecting and analyzing RNA molecules.

Traditional techniques used to study gene expression include Northern blotting, RNase protection, in situ hybridization, dot blots, and S1 nuclease assays. However, because coupled reverse transcription and PCR amplification (RT-PCR) is an easy, sensitive, and versatile technique, it is the method of choice for analyzing RNA. RT-PCR can be used to determine the presence or absence of a transcript, to quantify expression levels, and to clone rare messages without needing to construct and screen cDNA libraries.

RT-PCR kits differ not only with respect to the RT and PCR enzymes employed, but also with the procedures whereby cDNA synthesis and PCR amplification are coupled. MMLV reverse transcriptase (MMLV-RT), AMV RT, and Tth DNA polymerase are most commonly used to perform cDNA synthesis.^1,2,3,4 Second-strand synthesis and DNA amplification can then be carried out using Taq or Tth DNA polymerase,^1,2,3,4 or more recently, Taq-based DNA polymerase blends. Applying these blends to RT-PCR can lead to improved sensitivity, higher product yields, and amplification of longer complex targets.⁵ To date, little attention has been directed toward the accuracy of current RT-PCR procedures and the use of high-fidelity proofreading PCR enzymes. Although the frequency of mutations in the final product is thought to be influenced by the fidelity of both the RT and the DNA polymerase, the relative contributions of each enzyme are presently unknown.

Besides differences in enzymes employed, various procedures have been used for coupling cDNA synthesis and PCR amplification. RT-PCR is essentially carried out using either a two-step or a one-step procedure. In the two-step technique, cDNA synthesis is performed by the RT under optimal conditions, followed by PCR amplification with an appropriate thermostable DNA polymerase. The reaction tube must be opened after cDNA synthesis. PCR reagents are then added to the reaction tube (known as two-step/one-tube procedures) or the synthesized cDNA is transferred to a second tube for the PCR portion of the procedure. Stratagenes ProSTAR Ultra HF RT-PCR system (described below) uses the latter approach, called a two-step/two-tube procedure. Two-tube techniques are particularly useful when amplifying multiple targets from a single cDNA synthesis reaction.

In one-step RT-PCR procedures, cDNA synthesis and PCR amplification are performed in the same tube in a common buffer; there is no need to add reagents between the cDNA synthesis and PCR steps. One-step techniques are convenient, fast, and reduce the risk of sample contamination; they are particularly useful when amplifying the same target from multiple RNA samples. Only a few commercial one-tube RT-PCR kits are truly one-step kits, such as Stratagenes ProSTAR HF Single-Tube RT-PCR system. Other kits marketed as single tube/single buffer RT-PCR kits are actually two-step kits since they require additional cumbersome manipulations (e.g., buffer additions and the use of wax barriers).

RT-PCR Accuracy Is Influenced by DNA Polymerase Fidelity

The frequency of mutations in RT-PCR products is thought to be influenced by the replication fidelity of both the RT and the DNA polymerase, under the particular reaction conditions employed (e.g., number of duplications, pH, and [Mg²⁺]). The mutation frequency (M.F.) of an RNA amplification procedure can be estimated from the RT error rate, the DNA polymerase error rate, the number of cDNA template duplications, and the amplicon size. For example, consider the situation when an RNA template is reverse transcribed with MMLV-RT (error rate of 3.3 x10^-5/base⁶), and then a 1-kb portion is PCR amplified for 20 duplications (10⁶-fold amplification) using Stratagenes PfuTurbo DNA polymerase (error rate of 1.3 x 10^-6 M.F./bp/duplication⁷). The percentage of 1-kb cDNAs that contain errors is calculated to be 3.3% (3.3 x 10^-5/base x 1000 bases), while an additional 2.6% of the final clones would contain mutations introduced by PfuTurbo DNA polymerase [(1.3 x 10^-6)(1000 bp)(20 duplications)]. In this example, the overall M.F. would be 5.9%. In contrast, if the same RT-PCR reaction was carried out with AMV-RT (error rate of 5.9 x 10^-5/base⁶) and Taq DNA polymerase (8 x 10^-6 M.F./bp/duplication⁷), 5.9% of 1-kb cDNAs would contain RT-generated errors, and an additional 16% of the final clones would contain Taq-generated mutations [(8.0 x 10^-6) x (1000 bp) x (20 duplications)]. The overall M.F. in this case would be 21.9%. Thus, despite the relatively high error rates associated with RTs, the accuracy of the PCR enzyme is expected to contribute to overall mutation frequency due to the exponential amplification of the cDNA template during the PCR portion of RT-PCR procedures.

Table 1
RT-PCR Fidelity

cDNA Amplification Fidelity^a

DNA Polymerase

Error Rate
(x 10^-6)^b

Blue Plaques
(Number of mutants)

Total
Plaques

Mutation Frequency (%)

Pfu

1.3

122

10,441

1.2

TaqPlus Long

7.6

411

12,086

3.4

Taq

8.0

653

12,008

5.6

The PCR fidelity assay was carried out^7,10 except that the lacIOlacZa target was amplified from the same cDNA synthesis reaction mixture using different PCR enzymes. For cDNA synthesis, MMLV-RT was used essentially as described in the ProSTAR Ultra HF RT-PCR system protocol using in vitro transcribed RNA.

^bmutation frequency/bp/duplication; error rates measured on DNA templates.^3,6

To demonstrate that DNA polymerase fidelity contributes significantly to the accuracy of RT-PCR, we measured the frequency of mutations produced when a cDNA synthesis reaction mixture was amplified using different PCR enzymes. We modified our PCR-based forward mutation assay⁷ such that the lacI target gene (1.9-kb lacIOlacZa fragment) was amplified from cDNA synthesized from in vitro transcribed mRNA, rather than from plasmid DNA. RT-PCR products containing lacI were cloned into lambda arms, and the percentage of lacI mutants was determined in a color-screening assay.^7,10 Mutation frequencies were lowest in amplifications carried out with Pfu DNA polymerase (Table 1). In contrast, Taq DNA polymerase and the TaqPlus Long DNA polymerase blend produced mutation frequencies that were 5- and 3-fold higher, respectively. Therefore, DNA polymerase fidelity contributes to the accuracy of RNA amplification and should be an important consideration when choosing a PCR enzyme for RT-PCR procedures, particularly when the product is to be cloned, sequenced, and/or expressed.

Stratagenes Systems Include High-Fidelity PCR Enzymes

The ProSTAR RT-PCR systems features Stratagenes high performance/high fidelity PCR enzymes, PfuTurbo DNA polymerase and TaqPlus Precision DNA polymerase blend. PfuTurbo DNA polymerase, a blend of cloned Pfu DNA polymerase and a novel thermostable PCR enhancing factor, amplifies PCR products in higher yield than Taq DNA polymerase,⁸ and other proofreading single-enzyme formulations.⁹ Moreover, Pfu and PfuTurbo DNA polymerases exhibit significantly greater fidelity than all other PCR enzymes and DNA polymerase blends.^7,9 Stratagenes TaqPlus Precision PCR system is a Taq and Pfu DNA polymerase mixture, which has been specially formulated to achieve the highest replication accuracy possible for a Taq-based blend. TaqPlus Precision DNA polymerase exhibits a lower error rate than Taq and other commercially-available Taq-based DNA polymerase mixtures.^10,11

prostar Ultra HF RT-PCR System

Stratagenes ProSTAR RT-PCR systems use Moloney murine leukemia virus RT (MMLV-RT) for cDNA synthesis of specific target RNAs. The ProSTAR Ultra HF RT-PCR system includes all reagents necessary to carry out cDNA synthesis [MMLV-RT, first-strand buffer, dNTPs, oligo(dT), random primers, and RNase-free water] using total RNA or poly(A)⁺ RNA. The kit also includes all reagents necessary for subsequent, ultra high-fidelity PCR amplification with PfuTurbo DNA polymerase (PfuTurbo, 10X Ultra-HF PCR buffer, dNTPs). In this system, the cDNA synthesis reagents and protocol provide optimal performance using reaction volumes ranging from 10 to 50 l. Because the kits protocol is versatile, investigators can conserve RNA and cDNA synthesis reagents when a limited number of PCRs are performed (e.g., 10-l volumes) or can scale up the reverse transcription reaction (e.g., 50-l volumes) when multiple targets are amplified from the same cDNA synthesis reaction.

Figure 1

The ProSTAR Ultra HF RT-PCR system has been used to accurately amplify a broad range of target sizes from a variety of RNA sources, including human, mouse, yeast, and bacteriophage total RNA or poly(A)⁺-selected mRNA. High product yields were achieved for targets ranging in length from 270 bp to 7.6 kb (Figure 1, Panels A and B). In addition to long target capability, the ProSTAR Ultra HF RT-PCR system exhibits high sensitivity. RT-PCR products can be amplified from as little as 1 fg (low complexity; Figure 2) or 10 pg (high complexity; data not shown) of reverse-transcribed total RNA. Even low-abundance transcripts can be detected, as shown by the successful amplification of 1.8- to 7.6-kb fragments of human dystrophin from reverse-transcribed total RNA (200 ng of input RNA) (Figure 1).

Figure 2

ProSTAR HF Single-Tube RT-PCR System

The ProSTAR HF Single-Tube RT-PCR system includes all reagents necessary to carry out cDNA synthesis and high-fidelity PCR amplification in a convenient one-tube format (MMLV-RT, TaqPlus Precision DNA polymerase, HF RT-PCR buffer, dNTPs, and RNase-free water). Simply combine reagents into one tube along with gene-specific primers; cDNA synthesis and PCR amplification take place successively during an uninterrupted thermal-cycling program.

Figure 3

With this system, accurately amplify a broad range of target sizes can be accurately amplified from a variety of RNA sources (Figure 3). The kits TaqPlus Precision DNA polymerase blend permits high product yields for amplifications of both relatively short targets (300 to 550 bps; Figure 3, Panel A) and long targets from low-abundance message (1.8- to 5.9-kb dystrophin fragments; Figure 3, Panel B, Lanes 2 to 4). To demonstrate the systems sensitivity, a 325-bp RT-PCR product was amplified from as few as 10 molecules of an in vitro transcribed RNA (Figure 4). In addition, as little as 50 pg of total RNA was sufficient for amplifying the 1.8-kb dystrophin fragment from total RNA (low-abundance message; data not shown).

Figure 4

Both the ProSTAR systems also include test mRNA and a control primer set to verify reagent quality and RT-PCR procedures.

Conclusions

With Stratagenes ProSTAR RT-PCR systems, accurate cDNA amplification, high sensitivity, high product yield, and long-target capability are attainable. Use one of the two convenient kit formats, the Ultra HF RT-PCR system or the HF One-Tube RT-PCR system, to fulfill a variety of RNA amplification applications, including cDNA cloning and RNA detection/analysis.

Methods

RT-PCR reactions were carried out using the recommended protocols for the ProSTAR Ultra HF and HF Single-Tube RT-PCR systems. For the ProSTAR HF Single-Tube RT-PCR system, 50-l RT-PCR reaction mixtures contained 1X HF RT-PCR buffer, 200 M each dNTP, forward and reverse gene-specific primers (2 ng/l of each), RNA (200 ng of total RNA or 1 ng of bacteriophage MS2 RNA), 1.25 U of MMLV-RT, and 2.5 U of TaqPlus Precision DNA polymerase. Amplifications were carried out in Stratagenes RoboCycler Gradient 96 temperature cycler fitted with a Hot Top assembly, using 200-l thin-walled PCR tubes. The temperature cycling parameters for all targets incorporated the following: For cDNA synthesis, one cycle at 37C for 15 minutes (targets <2 kb) or 30 minutes (targets >2 kb); for PCR, one cycle at 95C for 1 minute; 40 cycles of 95C for 1 minute, 60C for 1 minute, and 68C for 2 minutes (targets <2 kb) or 2 minutes/kb (targets >2 kb); and one final extension cycle at 68C for 10 minutes.

For the ProSTAR Ultra HF RT-PCR system, 10-l cDNA synthesis reactions contained 1X MMLV-RT buffer, 1 mM each dNTP, 10 ng/l of oligo(dT), and RNA (200 ng of total RNA or 1 ng of bacteriophage MS2 RNA). The reaction mixtures were incubated at 65C for 5 minutes, then cooled to room temperature to allow the primers to anneal to the RNA. A total of 10 U of MMLV-RT was added, and cDNA synthesis was carried out at 37C for 15 minutes (targets <2 kb) or 30 minutes (targets >2 kb). PCR amplification reactions (50 l) contained 1X Ultra-HF PCR buffer, 200 M each dNTP, forward and reverse gene-specific primers (2 ng/l of each), 1 l of cDNA, and 2.5 U of PfuTurbo DNA polymerase. PCR amplification was performed as described above, except that extension at 68C was carried out for 3 minutes/kb.

RT-PCR products were electrophoresed on 0.8 to 1% agarose/1X TBE gels, stained with ethidium bromide, and imaged using the Eagle Eye II still video system. Lanes labeled M contain the Lambda/Hind III-fX174/Hae III DNA markers.

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* U.S. Patent No. 5,545,552 and patents pending
** U.S. Patent No. 5,556,772 and patents pending