Reverse transcriptases are necessary for manufacturing complementary DNA (cDNA) strands from RNA templates. Consequently, a deeper expertise of these enzymes’ attributes and also their implications in reverse transcription is an important for success in molecule biology experiments.

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Reverse transcriptases space enzymes created of distinctive domains the exhibit different biochemical activities. RNA-dependent DNA polymerase activity and RNase H activity are the predominant attributes of reverse transcriptases, although depending on the resource organisms there space variations in functions, including, because that example, DNA-dependent DNA polymerase activity. As displayed in Figure 1, the turning back transcription procedure typically involves a number of steps:

In the visibility of one annealed primer, reverse transcriptase binding to one RNA template and initiates the reaction.RNA-dependent DNA polymerase task synthesizes the security DNA (cDNA) strand, incorporating dNTPs.RNase H task degrades the RNA layout of the DNA:RNA complex.DNA-dependent DNA polymerase activity (if present) recognizes the single-stranded cDNA as a template, provides an RNA fragment together a primer, and synthesizes the second-strand cDNA.Double-stranded cDNA is formed.

(Learn more about reverse transcription reaction setup.)


Reverse transcription process
Figure 1. Reverse transcription process.
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RNase H activity


As discussed above, among the intrinsic properties of turning back transcriptases is the RNase H activity, which cleaves the RNA template of the RNA:cDNA hybrid at the same time with polymerization <1> (Figure 2). The RNase H task is undesirable for synthesis of long cDNAs because the RNA template may be degraded prior to completion that full-length turning back transcription. The RNase H activity may likewise lower reverse transcription efficiency, presumably due to its competition v the polymerase task of the enzyme.


RNase H activity of reverse transcriptases on cDNA synthesis
To boost cDNA synthesis, the RNase H task of reverse transcriptases has actually been diminished or decreased by introducing mutations right into the RNase H domain the the enzyme. Such mutations often result in greater yield and synthesis of much longer cDNAs (Figure 3) <2>.


Effects of RNase H task on cDNA synthesis
Figure 3. Effects of RNase H activity on cDNA synthesis. mRNAs of 9.5 kb, 7.5 kb, and also 5.2 kb to be reverse-transcribed in duplicate reactions, using reverse transcriptases v or there is no RNase H activity. The results demonstrate that the RNase H– turning back transcriptase was an ext efficient for producing full-length cDNA. M = Marker.


The capacity of a reverse transcriptase to withstand high temperatures is vital aspect the cDNA synthesis. Elevated reaction temperatures aid denature RNA with strong secondary structures and/or high GC content, enabling reverse transcriptases to check out through the sequence. Together a result, reverse warrior at greater temperatures allows full-length cDNA synthesis and higher yields, which leads to much better representation of an RNA populace by the cDNAs <3,4>.

Wild-type AMV turning back transcriptase displays higher thermostability than wild-type MMLV turning back transcriptase, with their optimal temperatures at 42–48°C and 37°C, respectively. Some engineered MMLV turning back transcriptases are modified to withstand temperatures up to 55°C v no discernible results on reverse transcription effectiveness (Figure 4). Such very thermostable reverse transcriptases are especially suitable to synthesize cDNA native GC-rich RNA templates. (White paper:Engineered reverse transcriptase)


Reverse warrior at elevated temperatures
Figure 4. Reverse warrior at elevated temperatures. RNA templates of varying lengths to be reverse-transcribed at different temperatures, making use of a highly thermostable, engineered MMLV reverse transriptase. RNA templates were removed by NaOH treatment, the result cDNAs were analyzed by denaturating agarose gelatin electrophoresis, and also reaction commodities visualized with the Invitrogen SYBR Gold main point Acid gelatin Stain. The results indicate thermostability the the enzyme through 100% task even at 56.4°C.


With gene-specific primers in one-step RT-PCR, reverse warrior at higher temperatures boosts specificity that the primers’ binding come the target. This strategy allows increased yield and also reduced background in subsequent PCR (Figure 5), making reverse transcriptases through high thermostability desirable for cDNA synthesis. (White paper:Improved one-step RT-PCR system)


Reverse transcription at one elevated temperature boosts PCR specificity
Figure 5. Reverse warrior at an elevated temperature boosts PCR specificity. MRNA because that four various genes was reverse-transcribed at different temperatures making use of oligo (dT)20 (12.3 kb mRNA) or gene-specific primers (9.3 kb, 7 kb, and also 5.5 kb mRNAs). Reverse transcription at 55°C generated greater specificity that the preferred targets in RT-PCR.


The processivity the a reverse transcriptase refers to the variety of nucleotides integrated in a solitary binding event of the enzyme. Therefore, a very processive turning back transcriptase deserve to synthesize longer cDNA strands in a much shorter reaction time (Figure 6). Some engineered MMLV turning back transcriptases can include as countless as 1,500 nucleotides in a solitary binding event, which to represent a processivity the is about 65 times greater than the of wild-type MMLV reverse transcriptase <5>.


Enzyme processivity is additionally associated through its affinity for the template. Together such, reverse transcriptases through high processivity room resistant to typical inhibitors that may have lugged over native the RNA sources. Examples of reverse transcriptase inhibitors encompass heparin and also bile salts from blood and also stool, humic acid and polyphenols native soil and plants, and also formalin and also paraffin native formalin-fixed, paraffin-embedded (FFPE) samples. These inhibitors regularly remain bound to RNA and/or reduce polymerization task <6>, and highly processive reverse transcriptases are far better able to get over such inhibition (Figure 7). (App notes:Improved RT-qPCR analyses of plant samples and also whole-blood RNA samples)


Learn around reverse transcriptase inhibitors, your inhibitory mechanisms, and also tips on overcoming inhibitors in turning back transcription.


Highly processive turning back transcriptases additionally perform better with RNA samples of low quality and also quantity <7>. This attribute makes extremely processive reverse transcriptases best for RNA isolated indigenous plant and also animal tissues and clinical research samples, which tend to it is in degraded as result of processing and also RNase-rich environments. Likewise, these enzymes space a great choice for experiments when minimal amounts the RNA space available.


Figure 7. Power of very processive turning back transcriptases in synthesis of cDNA from RNA samples with inhibitors or degraded RNA. (A) Reverse warrior was carried out through RNA ladders spiked with common enzyme inhibitors of organic sources (e.g., formalin indigenous FFPE samples, hematin and heparin from blood, bile salts from blood and also feces). Synthesized cDNA to be analyzed through alkaline gelatin electrophoresis making use of Invitrogen SYBR Gold nucleic Acid gelatin Stain. The turning back transcriptase v high processivity (H) displays greater efficiency in cDNA synthesis than enzymes v low processivity (L1–L4). (B) total RNA purified from various plant sources was assessed for integrity by gel electrophoresis. High-quality RNA (RIN >8) shows unique rRNA bands, conversely, degraded RNA (RIN 1–3) mainly contained smears and/or smaller RNA fragments. The degraded RNA to be reverse-transcribed v random hexamers, using reverse transcriptases that high processivity (H) or short processivity (L3, L4). QPCR to be performed v the resulting cDNA, for particular gene targets. The RT performance was determined by normalizing every Ct values to the Ct worths of the reverse transcriptase v high processivity. H = high processivity, together = short processivity, RIN = RNA truth Number.

The fidelity of turning back transcriptase represents succession accuracy maintained by the enzyme during synthesis the DNA from RNA. Fidelity is inversely correlated to one error price of reverse transcription. MMLV-based turning back transcriptases are reported to have an error price in the range of one in 15,000 come 27,000 nucleotides synthesized, v AMV reverse transcriptase displaying an even greater error rate <8-10>.

Reverse transcriptase’s fidelity might play a significant role in applications such as RNA sequencing whereby sequence accuracy is critical. For most other cDNA applications, the number of incorporated errors during reverse warrior is likely negligible for two reasons: the bulk of genes are much shorter than 10 kb and also the turning back transcription process does not amplify introduced errors in the cDNA.


Reverse transcriptases may display screen terminal nucleotidyl transferase (TdT) activity, which results in non–template-directed enhancement of nucleotides to the 3′ finish of the synthesized DNA. TdT task occurs only once the reverse transcriptase get the 5′ finish of the RNA template, to add 1–3 extra nucleotides to the cDNA end, and also exhibits specificity in the direction of double-stranded nucleic mountain substrates (e.g., DNA:RNA in the first-strand cDNA synthesis and DNA:DNA in the second-strand cDNA synthesis). In general, this intrinsic task is undesirable because the added nucleotides do not exchange mail to the template. The non–template-directed nucleotide addition commonly occurs through the choice in the bespeak of A>G≥C>T <9,11>.

Different reverse transcriptases own varying levels of intrinsic TdT activity. Through wild-type MMLV and AMV reverse transcriptases, 25–90% that synthesized DNA strands might contain extra nucleotides at the 3′ end. Engineered MMLV turning back transcriptases, ~ above the various other hand, often tend to display reduced intrinsic TdT activity. In enhancement to turning back transcriptase choice, the price of nucleotide enhancement depends top top reaction conditions such as the RNA:enzyme ratio, enzyme amount, incubation time, and also reaction temperature <9>.

For details applications such as full-length cDNA cloning, rapid amplification of cDNA end (RACE), and also RNA sequencing (RNA-Seq), turning back transcriptases might be induced to intentionally add a wire of Cs come the 3′ end of the cDNA. This kind of TdT task can be prompted during, or in a later on phase of, cDNA synthetic by high concentrations of magnesium and/or manganese ion <12,13>. An unified with a specially draft DNA oligo v a string of 3′ Gs (called a template-switching oligo), TdT activity can especially modify the 3′ cDNA end and also the 5′ RNA finish (Figure 8). Instances of these sequence changes include the development of a restriction website for subsequent cDNA cloning actions and/or enhancement of adapters because that downstream RNA sequencing measures <14-16>.


Figure 8. Nontemplate-dependent nucleotide addition by turning back transcriptase to modify the 3′ cDNA end.
As debated in this section, the endogenous properties of reverse transcriptases have the right to be exploited and modulated for effective cDNA-based experiments. In enhancement to opened up the research right into their aboriginal roles, including hereditary diversity and also retroviral replication, turning back transcriptases prove to be necessary tools because that molecular biologist for various applications prefer gene expression analysis and cDNA sequencing.

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Invitrogen Corp. (2003) thermal stability and also cDNA synthesis capability of SuperScript III turning back transcriptase. Focus 25(1): 19–24.