High fidelity reverse transcriptase (RT)
Higher thermostability, processivity, and fidelity than retroviral reverse transcriptases
Enzyme Properties and Novel Activity:
- Higher thermostability, processivity, and fidelity than retroviral reverse transcriptases, allowing full-length, end-to-end cDNA synthesis from highly structured or heavily modified RNAs (e.g., tRNAs), and RNAs containing GC-rich repeat expansions.
- Novel end-to-end template-switching activity that enables attachment of RNA-seq or PCR adapters during reverse transcription and eliminates the need for a separate RNA 3'-adapter ligation step.1 This template-switching activity greatly facilitates strand-specific RNA-seq library construction with less bias than procedures employing random hexamer primer or using RNA ligase for adapter ligation.
- Efficient cDNA synthesis from annealed primers. The annealed primer should have a predicted Tm of >60oC. Pre-incubation of enzyme with substrate in reaction mix for 30 min at room temperature and initiation of reaction by addition of dNTPs are recommended. Optimal conditions for new applications should be determined by testing a range of salt concentrations from 25 to 450 mM NaCl.
Advantages of the Enzyme:
1. Comprehensive strand-specific transcriptome profiling.
TGIRT®-seq of ribodepleted, fragmented Universal Human Reference RNA samples recapitulates the relative abundance of human transcripts and spike-ins comparably to non-strand-specific TruSeq v2 and better than strand-specific Tru-Seq v3. TGIRT®-seq is significantly more strand-specific than TruSeq v3 and eliminates sampling biases from random hexamer priming that are inherent to TruSeq. TGIRT®-seq shows more uniform 5' to 3' gene coverage and identifies more splice junctions than TruSeq. TGIRT®-seq enables simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq as structured small ncRNAs, including tRNAs, which are essentially absent from TruSeq datasets.
2. RNA-seq of whole-cell, exosomal, plasma, and other extracellular RNAs.
Fast processing time (<5 h for RNA-seq library construction through the PCR step); requires small amounts of RNA (low ng range); comprehensive transcript profiles including mRNAs and lncRNAs together with small ncRNAs, including full-length reads of tRNAs, pre-miRNAs, and other structured small ncRNAs; less bias and greater strand specificity than conventional methods.
3. RNA-seq library construction via TGIRT® template-switching in methods like RIP-seq, HITS-CLIP, irCLIP, CRAC, ribosome profiling.
Fast processing time (< 5 h for RNA-seq library construction through the PCR step); requires small amounts of RNA (low ng range); does not require RNA ligase, is less biased and more efficient by having fewer steps in the procedure.
4. Higher thermostability, processivity and strand-displacement activity than retroviral RTs.
Enables construction of RNA-seq libraries of polyadenylated RNAs using an anchored oligo(dT) primer with more uniform 5' to 3' coverage than retroviral RTs without a ribodepletion step.
Enables RNA-structure mapping via capillary electrophoresis-based methods like SHAPE or DMS structure mapping with significantly longer read lengths and fewer premature stops than for retroviral RTs.
Enables analysis of RNA templates containing GC-rich repeat expansions.
Enables synthesis of full-length, end-to-end cDNAs from tRNAs and other small structured/modified ncRNAs, which are refractory to retroviral RTs.
5. ssDNA-seq of human plasma and E. coli genomic DNAs.
Captures precise DNA ends with a simpler workflow by initiating DNA synthesis directly at the 3′ end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin.