Retroviral reverse transcriptases commonly used for cDNA synthesis exhibit a higher error rate than other DNA polymerases used in nucleic acid analysis techniques. This lack in accuracy leads to a significant number of base exchanges or frameshifts, which are further propagated in subsequent PCR reactions. High fidelity (proofreading) PCR enzymes have been available for many years; Roche′s new high accuracy reverse transcriptase is further able to synthesize high yields of full-length cDNA.
High mutation rate is a hallmark of retrovirus replication. This originates in the mechanism of genome replication by the viral-encoded reverse transcriptase, which converts the genomic RNA of the virus to a dsDNA. During this process, reverse transcription produces frequent replication errors. One accepted explanation of this inaccuracy is the lack of RT 3′-5′ exonuclease activity. The naturally high error rate of reverse transcriptases is not optimal for many different applications.
Transcriptor High Fidelity Reverse Transcriptase enzyme blend for high fidelity two-step RT-PCR of RNA up to 14 kb.
The core component of the Transcriptor High Fidelity Synthesis cDNA Synthesis kit is the Transcriptor High Fidelity Reverse Transcriptase, a blend of a recombinant reverse transcriptase and a proofreading mediating enzyme. The synergy between both enzymes is the key to the ability of the enzyme blend to reverse transcribe RNA templates with 7-fold higher fidelity compared to other commonly used reverse transcriptases.
The Transcriptor High Fidelity Reverse Transcriptase enzyme blend efficiently reverse transcribes templates up to 14 kb. Due to the high thermostability of both enzyme components and the specially optimized buffer system, reverse transcription is possible at temperatures up to +55°C. This allows the reverse transcription of GC-rich templates with high secondary structure, without the need to include additives that may negatively influence the accuracy of the reverse transcription reaction.
The recombinant reverse transcriptase included in the enzyme blend is expressed in E. coli. The enzyme has RNA-directed DNA polymerase activity, DNA-dependent DNA polymerase activity, unwinding activity and RNase H activity that degrades RNA in RNA:DNA hybrids. The latter circumvents the need to perform an additional time-consuming RNase H incubation step after reverse transcription, reducing reaction time and costs.
The Transcriptor High Fidelity cDNA Synthesis Kit provides all reagents required for first-strand cDNA synthesis reactions. For priming, three different primer systems can be used. Two cDNA synthesis primers are provided with the kit: random hexamer primers and an anchored-oligo(dT)18 primer. The latter is designed to bind at the beginning of the poly(A) tail to generate full-length cDNAs and to prevent priming from internal sites of the poly(A) tail. The 5′ ends of long mRNAs are often underrepresented, therefore this priming method is preferred for most applications. The use of random hexamer primers enables priming throughout the length of RNA for uniform representation of all RNA sequences and allows reverse transcription of RNAs that do not carry a poly(A) tail. The thermostable Protector RNase Inhibitor (included in the kit) protects RNA from degradation at high reaction temperatures.
Figure 1: Accuracy of the Transcriptor High Fidelity Reverse Transcriptase and a commonly used M-MuLV Reverse Transcriptase.
Error rate was determined by sequencing using the Genome Sequencer 20 System. RNA was reverse transcribed with the Transcriptor High Fidelity Reverse Transcriptase and a commonly used M-MuLV reverse transcriptase. After purification of the cDNA and amplification with a proofreading polymerase, the error rate of the reverse transcriptases was calculated by subtracting the error rate of the PCR control performed with plasmid DNA carrying the same sequence. The error rate of the Transcriptor High Fidelity ReverseTranscriptase is a mean value of four independent experiments in which at least 3.1 x 106 bases were sequenced. For the M-MuLV reverse transcriptase, 4.5 x 106 bases were sequenced.
Accuracy is represented as error rate -1, the average number of bases that are successfully reverse transcribed before a single transcription error is made. Transcriptor High Fidelity Reverse Transcriptase shows higher accuracy than a standard M-MuLV reverse transcriptase, resulting in lower error rate during cDNA synthesis.
Figure 2: Comparison of different reverse transcriptases for the reverse transcription of total RNA for different fragment sizes.
Total RNA (1 μg from human muscle total RNA for the 2.3 kb fragment; 1 μg from HeLa total RNA for the 5.4 kb and 9.4 kb fragments; 2 μg of rat brain total RNA for the 12.4 kb fragment) was reverse transcribed with different reverse transcriptases, according to the manufacturers′ recommendations. A 5 μl aliquot of each cDNA reaction was subsequently amplified with Roche′s Expand Long Range dNTPack. Results show that the Transcriptor High Fidelity cDNA Synthesis Kit efficiently transcribes a broad range of fragment sizes with greater yield and specificity compared to the reverse transcriptases from other suppliers.
Figure 3: Comparison of different incubation times for cDNA reactions, using the Transcriptor High Fidelity cDNA Synthesis Kit.
One microgram of HeLa total RNA was reverse transcribed at +50°C for the times indicated. Five-microliter aliquots of the cDNA reactions were amplified with primers for an 8.5 kb fragment, using Roche′s Expand Long Range dNTPack. All reactions were performed in duplicate. Results show that the Transcriptor High Fidelity cDNA Synthesis Kit efficiently transcribes RNA with high speed and accuracy.
Figure 4: Comparison of different incubation times for cDNA reactions, using the Transcriptor High Fidelity cDNA Synthesis Kit.
1 μg of HeLa total RNA was reverse transcribed at +50°C for the times indicated. 5 μl aliquots of the cDNA reactions were amplified with primers for an 8.5 kb fragment, using Roche′s Expand Long Range dNTPack. All reactions were performed in duplicate
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