AccelerRT® 5G Template Switching RT Enzyme Mix

Product Description

AccelerRT® 5G Template Switching RT Enzyme Mix (RNase H-) is a novel RT enzyme that was evolved in vitro from MMLV RT. The enzyme possesses RNA- and DNA-dependent polymerase activities but lacks RNase H activity. Its efficient template-switching function allows it to be used for full length cDNA products. The engineered enzyme has greatly improved thermal stability, processability and synthesis rates compared to the wild type MMLV RT enzyme.

Applications

• 5′-RACE (Rapid Amplification of cDNA Ends)
• First-strand cDNA synthesis for full length cDNA products
• Construction of single cell sequencing libraries
• Discovery and detection of fusion genes
• Generation of labeled cDNA probes
• RNA analysis by primer extension

Supporting Data

Template Switching Function

Figure 1. Introduction to the principle of the Template Switching RT Enzyme. The Oligo(dT) VN Primer is used to synthesize the first-strand cDNA. Upon reaching the 5′ end of the RNA template, a few non-template nucleotides are added to the 3′ end of cDNA (Template-switching) using the terminal deoxynucleotidyl transferase (TdT) activity of reverse transcriptase. The second-strand of the cDNA is synthesized using a template-switching oligo. Finally, the full-length cDNA product is obtained by PCR amplification using reverse gene-specific primers and forward Template-switching oligo (TSO)-specific primers.

High Efficiency of Template Switching Synthesis

Figure 2. AccelerRT® 5G template Switching reverse transcription reaction was performed using the TSO primer, followed by PCR amplification using the 5 ‘-end portion of the TSO primer and the gene-specific primer(A). The PCR amplification products were subjected to agarose gel electrophoresis(B) and the efficiency of template switch was calculated by product density value and product length (table).  

Figure 3. Template Switching Synthesis efficiency of AccelerRT® 5G Template Switching RT Enzyme Mix. 5G Template Switching reverse transcription reaction was performed using the TSO primer, followed by PCR amplification using the 5 ’-end portion of the TSO primer and the gene-specific primer. The PCR amplification products were subjected to agarose gel electrophoresis. SuperScript IV RTase (Thermo Fisher #18090050) was used for comparison.

Effective amplification of  cDNA Synthesis

Figure 4. Total RNA from Hela cells was used in a reverse transcription reaction with AccelerRT® 5G Template Switching RT Enzyme Mix. The synthesized cDNA was used as a template in subsequent PCR using the UltraHiPF® DNA Polymerase Kit.