Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction.

By York-Yuan Zhu, E. Machleder, A. Chenchik, R. Li, P. Siebert. BioTechniques, 2001

807 citations recorded by the source corpus.

Here, we describe a fast, simple method for constructing full-length cDNA libraries using SMART technology. This novel procedure uses the template-switching activity of Moloney murine leukemia virus (MMLV) reverse transcriptase to synthesize and anchor first-strand cDNA in one step. Following reverse transcription, three cycles of PCR are performed using a modified oligo(dT) primer and an anchor primer to enrich the cDNA population for full-length sequences. Starting with 1 microgram human skeletal muscle poly(A)+ RNA, a cDNA library was constructed that contained 3 x 10(6) independent clones with an average insert size of 2 kb. Sequence analysis of 172 randomly selected clones showed that 77% of cDNA clones corresponding to known genes contained intact open reading frames. The average length of complete open reading frames was 2.4 kb. Furthermore, 86% of the full-length clones retained longer 5' UTR sequences than the longest 5' end deposited in the GenBank database. cDNA libraries generated using this method will be useful for accelerating the collection of mRNA 5' end sequence information, which is currently very limited in GenBank.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/bf030d824a75f483dd0616d63ea1ab367caa399d

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

Citations in the corpus

Genome-wide mapping of alternative splicing in Arabidopsis thaliana. (2010, methodology): “Libraries were generated using the SMART method (Zhu et al. 2001). cDNA (5–7 mg in a 50-mL volume) was mixed with 750 mL of Illumina nebulization buffer and fragmented for 7 min in a nebulizer (Invitrogen) using compressed nitrogen at 35 psi.”
Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes (2009, methodology): “First strand cDNA was synthesized from mRNA using SMART technology [91] as part of the SMART cDNA Library Construction kit (Clontech; Mountainview, CA, USA).”
Exponential scaling of single-cell RNA-seq in the past decade (2017, methodology): “This method is based on the intrinsic ability of the transcriptases of the Moloney murine leukaemia virus (MMLV) to add a small number of nucleotides (mostly cytosines) when the RT reaches the end of the mRNA (Zhu et al., 2001).”
Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing (2008, methodology): “Complementary DNA was synthesized from the two RNA pools and normalized (Zhu et al. 2001; Shagin et al. 2002; Zhulidov et al. 2005; Evrogen: www.evrogen.com) in order to prevent over-representation of the most common transcripts (Fig.”
Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets (2015, methodology): “The mRNAs are then reversetranscribed in bulk, forming STAMPs, and template switching is used to introduce a PCR handle downstream of the synthesized cDNA (Zhu et al., 2001).”
Single-cell RNA-seq: advances and future challenges (2014, neutral): “This SMART-seq method ( S witching m echanism a t 5 end of the R NA t ranscript) utilizes an intrinsic property of RT M-MuLV to add three to four cytosines specifically to the 3 end of the first cDNA strand, which is subsequently used to anchor a universal PCR primer (117).”
CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq (2016, methodology): “The STRT method introduced early barcoding at the reverse transcription stage [2], thereby enabling highlymultiplexed analyses, and adapted a template switching mechanism based on the ability of the reverse transcriptase to tag the end of the cDNA [3], eliminating the need for the polyadenylation re”