Biotechnology Letters 22: 1775–1778, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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A formulation of multiple reverse transcriptases for improved RT-PCR Claire L. Nevett∗ & Ariel Louwrier Advanced Biotechnologies Ltd., ABgene House, Blenheim Road, Epsom, Surrey KT19 9AP, UK ∗ Author for correspondence (Fax: +01372 741414; E-mail:
[email protected]) Received 19 July 2000; Revisions requested 9 August 2000; Revisions received 6 September 2000; Accepted 7 September 2000
Key words: avian myeloblastosis virus, Moloney murine leukaemia virus, polymerase chain reaction, reverse transcriptase
Abstract A formulation of reverse transcriptases comprising enzymes derived from avian myeloblastosis virus and from Moloney murine leukaemia virus has provided an improved method for amplifying nucleic acid sequences by coupled reverse transcription and polymerase chain reaction. The properties of each enzyme appear to complement the other and demonstrate enhanced sensitivity and increased product yield in comparison to either enzyme used separately.
Introduction Gene expression is a fundamental cellular process that differs from cell to cell and is dependent on many factors including cellular environment or state of differentiation. Consequently, the measurement of RNA is key to understanding normal cell function, dysfunction causing disease and the influence of changes in extracellular environment. Coupled reverse transcription and polymerase chain reaction (RT-PCR) is a commonly used technique, providing high sensitivity RNA detection suitable for the analysis of low copy number mRNAs. Clearly, the product yield of the RT-PCR reaction is largely dependent on the success of reverse transcription, since the level of amplification achievable by PCR is dependent on the quantity of single-stranded DNA starting material. Therefore, consideration of the requirements of this step is crucial to achieving high yields of end product. Traditionally, reverse transcriptases have been used singly to catalyse first strand synthesis, with those derived from avian myeloblastosis virus (AMV) or Moloney murine leukaemia virus (MMuLV) being common enzymes of choice. The two enzymes are characterised by distinct RNase H activities and optimum catalytic temperatures. AMV possesses higher activities of RNase H relative to MMuLV (Verma
1975), whilst the latter has a lower optimal reaction temperature than the former (Fuchs et al. 1999, Sambrook et al. 1989). The lower optimal temperature of MMuLV may favour formation of RNA secondary structure, reducing the efficiency of first strand synthesis. RNase H is responsible for degradation of RNA in RNA:DNA hybrids and its role in RT-PCR has been extensively studied (Gerard 1981). However, opinion remains divided as to the balance of its effects. Resultant RNA degradation is considered to be detrimental to successive rounds of RT, reducing the amount of template available. In contrast, it is believed to increase primer binding efficiency in the PCR step by removing the RNA, which may competitively inhibit primer-template interaction. To the best of our knowledge there is no information in the scientific literature addressing the use of multiple reverse transcriptases in RT-PCR. We aim to examine this issue and address the relevance of RNase H activity to it. To this end we have produced a unique, optimised blend of AMV and MMuLV, and have used this RT blend to investigate the potential for enhancing both the sensitivity and product yield of RT-PCR. Here we demonstrate that this combination of enzymes improves RT-PCR and conclude that the enhanced properties of the enzyme blend are the result
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Fig. 1. MS2 RNA was diluted serially in 10-fold steps from 1 ng to 1 fg. Each was added to a coupled RT-PCR reaction mixture containing MS2 primers and either 0.1 U avian myeloblastosis virus reverse transcriptase (AMV) (a), 0.5 U AMV (b), 0.4 U Moloney murine leukemia virus reverse transcriptase (MMuLV) (c), 0.5 U MMuLV (d) or 0.1 U AMV + 0.4 U MMuLV (e).
of a fine balance between the greater thermal stability of AMV and the lower RNase H activity of MMuLV.
Fig. 2. Human testicular total RNA was diluted serially in 10-fold steps from 0.1 µg to 10 pg. Each was added to a coupled RT-PCR reaction mixture containing β-actin primers and either 0.1 U avian myeloblastosis virus reverse transcriptase (AMV) (a), 0.5 U AMV (b), 0.4 U Moloney murine leukemia virus reverse transcriptase (MMuLV) (c), 0.5 U MMuLV (d) or 0.1 U AMV + 0.4 U MMuLV (e).
0.4 U MMuLV containing a point mutation that eradicated RNase H activity (RNase H− ) (GeneSys) under conditions described above.
Materials and methods MS2 viral RNA (Boehringer Mannheim) and human testicular total RNA (Clontech) were serially diluted in 10-fold steps from 1 ng to 1 fg and from 0.1 µg to 10 pg, respectively. Each of these dilutions was added to 50 µl of One-Step RT-PCR mix (ABgene), and either 10 pmol MS2 primers or 25 pmol β-actin primers. To each of these reactions 0.1 U AMV, 0.5 U AMV, 0.4 U MMuLV, 0.5 U MMuLV or 0.1 + 0.4 U of AMV + MMuLV blend were added. The reaction mixtures were then subjected to RT-PCR under the following conditions: 47 ◦ C, 30 min; 94 ◦ C, 2 min; 40 cycles of 94 ◦ C, 20 s; 60 ◦ C, 30 s; 72 ◦ C, 40 s; and 72 ◦ C, 5 min, unless stated otherwise. The products were separated by Tris/acetate/EDTA (TAE) agarose gel electrophoresis on a 1% (w/v) agarose gel, the gel stained with ethidium bromide, and DNA products visualised using a UV transiluminator. RT-PCR was also performed using a combination of 0.1 U AMV +
Results and discussion Consideration of the different catalytic properties of AMV and MMuLV, including both temperature optima and RNase H activity, lead to the proposal that their combination in RT-PCR might, under certain conditions, maximise the beneficial effects on product formation. The optimal catalytic temperatures for each of the two reverse transcriptases are quite disparate: AMV: 48–55 ◦ C; MMuLV: 37–42 ◦ C (Wilkinson 1988). A suitable reaction temperature for a blend of the two enzymes is therefore likely to lie between the optimal temperatures of each enzyme. A range of reaction temperatures was analysed, and the optimum temperature for the enzyme blend was determined to be 47 ◦ C under model assay conditions (MS2 RT-PCR).
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Fig. 3. MS2 RNA was diluted serially in 10-fold steps from 1 ng to 1 fg. Each was added to a 50 µl coupled RT-PCR reaction mixture containing MS2 primers and either 0.1 U avian myeloblastosis virus reverse transcriptase (AMV), 0.4 U Moloney murine leukemia virus reverse transcriptase MMuLV), or 0.1 U AMV + 0.4 U MMuLV (blend). Reverse transcription was performed using AMV at 47 or 50 ◦ C; using MMuLV at 42 or 47 ◦ C and using the AMV + MMuLV blend at 42, 47 or 50 ◦ C, whilst all other reaction conditions were as described.
A range of enzyme concentration ratios was examined, and optimal product formation was achieved using a combination of 0.1 U AMV and 0.4 U MMuLV. In order to establish if the use of this enzyme blend was superior to the use of the individual enzymes separately, two important criteria needed to be met. The sensitivity and product yield of the enzyme blend should be superior to those of the individual enzymes, present either in amounts equal to those in the blend, or amounts equivalent to the total combined reverse transcriptase activity in the blend, i.e., 0.1 U or 0.5 U for AMV and 0.4 U and 0.5 U for MMuLV. Both the sensitivity and product yields of the blend were higher under the tested conditions (Figure 1). In order to assess any dependency on the RNA source (viral genomic RNA or total RNA) in these experiments and test the generality of the principal in this regard, the experiment was repeated using human total RNA. The results generated were similar (Figure 2), indicating that the RNA source played no detectable role. However, during these experiments the thermal conditions were kept constant. In order to remove any bias that might arise from the fact that the individual enzymes were being used at sub-optimal temperatures whilst being compared to the blend, further experiments were designed. Reactions were performed using equivalent amounts of enzyme to those in the blend
(0.1 U AMV and 0.4 U MMuLV) at their optimal temperatures (42 and 50 ◦ C for MMuLV and AMV respectively) and were compared to reactions using the blend (0.5 U) at 42, 47 or 50 ◦ C (Figure 3). The sensitivity obtained using the blend was greater than that of either enzyme separately, irrespective of the reaction temperature. Not surprisingly, the proportionality of product yield with respect to template concentration obtained by the blend was better at 47 ◦ C than at 42 or 50 ◦ C, confirming the difference in activities of both constituent enzymes at the different temperatures. As the enzymes were used at their optimised constituent blend concentrations, this last experiment was repeated using 0.5 U of each individual enzyme. No differences in sensitivity were recorded, indicating that this effect was not measurably concentrationdependant, although the issue of substrate limitation cannot be entirely eliminated. We examined next the role of the associated RNase H activity, as its presence would cause template degradation, hence limiting the sensitivity of the reaction. Now, the absence of this activity would maintain the concentration of the original RNA template rather than degrading and therefore reducing it. Furthermore, MMuLV constitutes 80% of the reverse transcriptase activity in the blend. This suggested that the removal of the vast majority of the RNase H activity could be
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Fig. 4. MS2 RNA was diluted serially in 10-fold steps from 1 ng to 1 fg. Each was added to a coupled RT-PCR reaction mixture containing 0.1 U avian myeloblastosis virus reverse transcriptase (AMV) + 0.4 U wild-type Moloney murine leukemia virus reverse transcriptase (MMuLV) (wt), or 0.1 U AMV + 0.4 U MMuLV containing a point mutation that eliminated RNase H activity (H− ).
achieved by introducing a MMuLV RNase H− mutant. If the RNase H activity was responsible for a lack in reaction sensitivity, its removal would dramatically increase the sensitivity of the blend, which proved to be the case (Figure 4). Use of the mutant MMuLV improved the sensitivity of RNA detection by two orders of magnitude in terms of template concentration. This series of deductive experiments lead us to conclude that the blend of AMV and MMuLV produces greater sensitivity and product yield in RT-PCR than either enzyme used individually, and that this can be further increased with the introduction of an RNase H− mutant of MMuLV in the blend of enzymes. These data may have particular impact on the detection of low abundance transcripts and on their quantitation.
References Fuchs B, Zhang K, Rock MG, Bolander ME, Sarkar G (1999) High temperature cDNA synthesis by AMV reverse transcriptase improves the specificity of PCR. Mol. Biotechnol. 12: 237–240. Gerard M (1981) Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase HI). Biochemistry 20: 265–265. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory Press. Verma IM (1975) Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. J. Virol. 15: 843–854. Wilkinson DA (1988) Getting the message with RT-PCR. A profile of kits for first strand synthesis and RT-PCR. Scientist 12: 20–22.
