Oligo Modifications
Support and Educational Content

Use of template switching oligos (TS oligos, TSOs) for efficient cDNA library construction

Quick facts

Composition: Chimeric DNA/RNA oligo

Ordering path: Order as a Custom RNA Oligo

Typical length: ~30 bases

Scales: 100 nmol to large scale synthesis

Purification: Standard desalt; RNase-free HPLC available on request

Website ordering symbol: Use lower case “r” in front to specify ribonucleotide bases (e.g,, ACTGCrGrGrG)

Underrepresentation of cDNA 5' ends

Emerging high-throughput technologies such as RNA-seq have enabled functional dissection of complex transcriptomes with a minimal amount of RNA input. Accurate quantification of individual transcripts or identification of unknown transcription start sites requires an efficient approach to convert mRNA molecules into full-length cDNA. However, conventional cDNA construction methods usually result in an underrepresentation of the 5’ ends of cDNA. This outcome poses a significant technical obstacle for researchers interested in transcriptome analysis in non-model organisms or in gene discovery [1,2].


SMART strategy and template switching oligo secure 5' end sequences

Described originally in 2001, a strategy frequently referred to as “SMART” (switching mechanism at the 5' end of the RNA transcript) has shown promise in generating full-length cDNA libraries, even from single-cell–derived RNA samples [1,3]. This strategy relies on the intrinsic properties of Moloney murine leukemia virus (MMLV) reverse transcriptase and the use of a unique template switching oligonucleotide (TS oligo, or TSO). During first-strand synthesis, upon reaching the 5’ end of the RNA template, the terminal transferase activity of the MMLV reverse transcriptase adds a few additional nucleotides (mostly deoxycytidine) to the 3' end of the newly synthesized cDNA strand. These bases function as a TS oligo-anchoring site. Upon base pairing between the TS oligo and the appended deoxycytidine stretch, the reverse transcriptase “switches” template strands, from cellular RNA to the TS oligo, and continues replication to the 5' end of the TS oligo. By doing so, the resulting cDNA contains the complete 5' end of the transcript, and universal sequences of choice are added to the reverse transcription product. Along with tagging of the cDNA 3' end by oligo dT primers, this approach makes it possible to efficiently amplify the entire full-length transcript pool in a completely sequence-independent manner [4]. See Figure 1 for a schematic of this method.

Figure 1. Generate full-length, double-stranded cDNA library construction through use of Oligo-dT30 VN and a template switching oligo (TS oligo, TSO).


TS oligo structure

The simple version of a TS oligo is a DNA oligo sequence that carries 3 riboguanosines (rGrGrG) at its 3' end [1]. The complementarity between these consecutive rG bases and the 3' dC extension of the cDNA molecule empowers the subsequent template switching [5]. A more recent study suggested replacing the 3' most rG with a locked nucleic acid base (LNA), possibly due to the enhanced thermostability of the LNA monomer, which would be advantageous for base pairing [6]. Interestingly, a systematic study was conducted in 2013 to compare the multiple modified TS oligo versions side-by-side, and the data indicated that RNA/DNA hybrids provide a superior level of 5' cap-specific enrichment, even though the DNA/LNA duplexes were expected to be more thermodynamically favored [7].

Driven by the growing interest in single-cell transcriptomics, alternative modification patterns have been explored to further improve TS oligo performance. For instance, in 2010 researchers reported an approach to reduce library background and thereby improve cDNA yield by incorporating isomeric nucleotides into the TS oligo [8]. In this study, 2 modified bases, iso-dC and iso-dG, were appended to the 5’ end of the TS oligo. These 2 modifications, which are chemical variants of cytosine and guanine, respectively, form hydrogen bonds with each other but not with naturally occurring C and G nucleotides. As anticipated, this modified version demonstrated efficacy in minimizing the concatenation of TS oligos, which typically results from cycles of reverse transcriptase activity [8,9].


Ordering TS oligos

Taken together, TS oligos attach a universal primer-binding site through the activity of MMLV reverse transcriptase, enabling exclusive amplification of intact cDNA molecules. TS oligos containing 3' rGs can be ordered from the Custom RNA Oligos ordering page on the IDT website. You can further customize your TS oligo with unique properties by incorporating additional chemical modifications from this same ordering page.

Contact us at applicationsupport@idtdna.com with any questions about ordering modified oligos or to discuss your experimental design with our scientific applications experts.


References

  1. Zhu YY, Machleder EM, et al. (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction Biotechniques, 30(4):892–897.
  2. Wellenreuther R, Schupp I, et al. (2004) SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones. BMC Genomics, 5(1):36.
  3. Ramskold D, Luo S, et al. (2012) Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol, 30(8):777–782.
  4. Shapiro E, Biezuner T, Linnarsson S. (2013) Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet, 14(9):618–630.
  5. Turchinovich A, Surowy H, et al. (2014) Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA. RNA Biol, 11(7):817–828.
  6. Picelli S, Faridani OR, et al. (2014) Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc, 9(1):171–181.
  7. Harbers M, Kato S, et al. (2013) Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparation. BMC Genomics, 14:665.
  8. Kapteyn J, He R, et al. (2010) Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples. BMC Genomics, 11:413.
  9. Saliba AE, Li L, et al. (2016) Single-cell RNA-seq ties macrophage polarization to growth rate of intracellular Salmonella. Nat Microbiol, 2:16206.

Product focus—oligos, modifications, dsDNA fragments

Custom oligonucleotides and primers

You can order up to 1 µmol of desalted, custom synthesized DNA oligonucleotides, and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on. Learn more or order now.

IDT has the expertise to deliver custom-synthesized RNA with the yield and purity that today's researcher demands. RNA oligos are available from 10–60 bases for 100 nmol, 5–60 bases for 250 nmol and 1 µmol, and 5–50 bases for 5 µmol and 10 µmol. Sequences from 60–120 bases are available as high-fidelityUltramer® RNA Oligonucleotides delivered at guaranteed quantities of 4, 20, or 80 nmol. All RNA oligos are available with a range of modifications. See the IDT modifications portfolio for more information.

Standard custom RNA is shipped deprotected and desalted in 2–3 business days or deprotected and purified in 4–6 business days. Please inquire for turnaround on 5 µmol and 10 µmol RNA synthesis. Learn more or order Custom RNA Oligos.


Oligo modifications

Review a list of the common modifications IDT can add to oligonucleotides here Not finding a modification you need on the IDT website? We will consider any modification you need. Just send your request to noncat@idtdna.com.


Custom dsDNA Fragments

Rather than annealing oligonucleotides to obtain dsDNA fragments, when your fragment size is 125 bp or longer, it might make more sense to order gBlocks® Gene Fragments. gBlocks Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.


Learn more about gBlocks Gene Fragments at www.idtdna.com/gBlocks.

Additional reading

Generate codon balanced libraries for mutagenesis with trimer modifications—Modification highlight: Incorporating oligo codon trimers into oligo libraries results in balanced encoding of amino acids and eliminates unwanted stop codons. Such oligo libraries are useful for mutagenesis experiments to prepare proteins for screening for potential improvements in biological function.

Insert an abasic site into your sequence—Modification highlight: Use the dSpacer, rSpacer, and Abasic II modifications to introduce abasic sites into DNA or RNA oligonucleotides. These modifications create a single base space that replicates the loss of base pairing ability by a nucleotide.

Increase the Tm of short, AT-rich primers and probes—Modification highlight: Add this modified base to increase the melting temperature (Tm) of primers and probes. It is especially useful when you need to work with short A-T rich sequences.


Review other DECODED Online newsletter articles on oligo handling and analysis and oligo modifications.


You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.


Author: Brian Wang, PhD, is the Genomic Tools Market Development Manager at IDT.

© 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


IDT web tools for oligo properties

Free, online tools for oligo design, secondary structure, dilution, and resuspension.

Try them now ≫


Related Articles

DNA Oligonucleotide Resuspension and Storage

Guidelines and recommendations for how to resuspend and store newly synthesized oligonucleotides.

Read more ≫

Calculation Tips for Resuspending and Diluting Nucleic Acids

Easy guidelines for making a 100 µM solution; calculating nmoles, µg, copy number, and concentration; and determining concentration equivalencies.

Read more ≫

Understanding Melting Temperature

Advice on considerations for better oligo design: oligo concentration, salt, and SNPs.

Read more ≫

Oligonucleotide Modifications: Choosing the Right Mod for Your Needs

Guidelines on selecting specific oligonucleotide modifications and how they can help you in your research.

Read more ≫

Which Biotin Modification to Use?

Applications of each of the different biotins available from IDT.

Read more ≫