AccelerRT® 5G Template Switching Reverse Transcriptase(RNase H-) is a novel RT enzyme that was developed through in vitro evolution of MMLV RT. The enzyme possesses an RNA and DNA-dependent polymerase activity but lacks of RNase H activity. Its efficient template switching function can be used for full length cDNA products. The engineered enzyme features greatly improved thermal stability, processability and increased synthesis rates compared to the wild type MMLV RT enzyme.
- 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.
- Analysis of RNA by primer extension.
Figure 1. Introduction to the principle of Template Switching Reverse Transcriptase. 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 using the terminal deoxynucleotidyl transferase (TdT) activity of reverse transcriptase (Template-switching).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 TSO-specific primers. 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. Broad thermal stability of AccelerRT® 5G Template Switching Reverse Transcriptase. 1ng Hela total RNA was reverse transcribed using AccelerRT® 5G Template Switching Reverse Transcriptase at different temperatures from 42 ℃ to 60℃. The GAPDH and B2M cDNAs were amplified with BlazeTaq™ SYBR® Green qPCR mix 2.0(Cat.# QP031). Figure 4. First-strand cDNA was generated from 100 ng to 1 pg of total RNA from Hela cells using AccelerRT® 5G Template Switching Reverse Transcriptase. The synthesized cDNA was used as a template for qPCR using BlazeTaq™ SYBR® Green qPCR mix 2.0(Cat.# QP031). Figure 5. RT efficiency of AccelerRT® 5G Template Switching Reverse Transcriptase was demonstrated using 1ng Hela total RNA reverse transcribed for 10min, 15min, 30min and 60min. The GAPDH and B2M cDNAs were amplified using BlazeTaq™ SYBR® Green qPCR mix 2.0(Cat.# QP031). Figure 6. Various inhibitors were added to total HeLa RNA, then was used in a reverse transcription reaction with AccelerRT® 5G Template Switching Reverse Transcriptase, The synthesized cDNA was used as a template in subsequent qPCR using BlazeTaq™ SYBR® Green qPCR mix 2.0(Cat.# QP031). Figure 7. Total RNA from Hela cells was used in a reverse transcription reaction with AccelerRT® 5G Template Switching Reverse Transcriptase. The synthesized cDNA was used as a template in subsequent PCR using the UltraHiPF™ DNA Polymerase Kit (Cat.# PC018). Figure 8. Total RNA from H2228 cells was used in a reverse transcription reaction with AccelerRT® 5G Template Switching Reverse Transcriptase. The synthesized cDNA was used as a template in subsequent PCR using the 5 '-end portion of the TSO primer and the gene-specific primer ALK gene(A). The amplification products in Figure A was verified by amplification with the EML4 forward primer and the ALK reverse primer(B). * IP protected and AccelerRT® 5G Trademark owned by GeneCopoeia, Inc.Template Switching function
High Efficiency of Template Switching Synthesis
Broad thermostability
Exceptional RNA template sensitivity
High efficiency of reverse transcription
Consistent performance in the presence of a variety of inhibitors
Effective amplification of cDNA Synthesis
Discovery and detection of fusion genes
