Because cDNA synthesis is susceptible to interruption by secondary structures in the template RNA, the 5' ends of genes are typically underrepresented in cDNAs synthesized by conventional methods. During SMART cDNA synthesis truncated products resulting from premature termination of the reverse transcription reaction do not incorporate the SMART(er) oligonucleotide and consequently are not amplified during PCR. Thus, cDNA created using our SMART technology and amplified by long-distance PCR is enriched for full-length cDNA. Furthermore, because the 5’ SMART(er) sequence and modified oligo dT primer are not added onto genomic DNA or cDNA transcribed from ribosomal RNA, cDNA that is generated using SMART is free of these contaminating agents.
One of the greatest advantages of SMART technology is its increased efficiency compared to traditional technologies such as adaptor ligation. Its high efficiency and sensitivity enables you to use a very limited quantity of starting material, such as microdissected tissues, laser-captured cells, biopsy samples, etc. As little as 1-2 ng of total RNA is sufficient for generating a highly representative cDNA pool for different downstream applications.
During first-strand SMARTer cDNA synthesis known universal primer sequences are incorporated at both ends of the cDNA. Following first-strand synthesis, SMARTer cDNA is immediately available for PCR amplification.
SMART (Switching Mechanism at 5’ End of RNA Template) is a unique technology that allows the efficient incorporation of known sequences at both ends of cDNA during first strand synthesis, without adaptor ligation. The presence of these known sequences is crucial for a number of downstream applications including amplification, RACE, and library construction. While a wide variety of technologies can be employed to take advantage of these known sequences, the simplicity and efficiency of the single-step SMART process permits unparalleled sensitivity and ensures that full-length cDNA is generated and amplified.
The SMART cDNA Library Construction Kit is designed for the cloning of full-length cDNA into a phage TriplEx2 vector. The kit combines the SMART technology (Switching Mechanism At 5' end of RNA Template) for cDNA amplification with adaptor-free, directional cloning into the TriplEx2 vector. This kit contains two separate protocols, allowing you to choose a method based on your starting material. The first protocol employs long-distance PCR (LD PCR), for researchers limited by their starting material. As little as 50 ng of total RNA can be used as starting material (1). The second protocol provides a more straightforward protocol for researchers with abundant amounts of starting material (i.e., 1 µg or more of poly A+ RNA). SMART libraries contain a higher percentage of full-length clones than libraries constructed by conventional methods or other full-length cDNA synthesis protocols. Thus, clones isolated from SMART cDNA libraries contain sequences corresponding to the complete 5' untranslated region of the mRNA (2).
SMART cDNA Synthesis Technology ensures uninterrupted cDNA synthesis, creating cDNAs with well-represented 5’ end sequences. Since terminal transferase activity and the subsequent SMART switching process occur preferentially at the 5' ends of eukaryotic mRNAs, truncated products resulting from premature termination of the reverse transcription reaction generally do not incorporate the SMART(er) oligonucleotide, and consequently are not amplified during PCR. Thus, cDNA pools created using our SMART technology and amplified by long-distance PCR are enriched for full-length cDNA. Additionally, because the 5’ SMART(er) sequence and modified oligo dT primer are not added onto genomic DNA or cDNA transcribed from ribosomal RNA, cDNA that is generated using SMART is free of these contaminating agents.
This optimized protocol significantly reduces non-specific background, essential when handling very small sample sizes.
Specific enrichment for 5 ends
We have designed a specialized SMART Oligo that preferentially hybridizes to 5 ends of the cDNA being synthesized.
The In-Fusion SMARTer Library Construction protocol can be completed in fewer steps than other library construction methods due to a highly efficient cDNA synthesis and cloning process. Only three enzymes are required to complete the entire protocol, as opposed to the usual 6–8 enzymes required for other methods. The SMART(er) cDNA synthesis protocol is user friendly and straightforward with no adaptor ligation or tailing steps. Your precious RNA is subjected to the to the least possible handling, thereby minimizing the risk of degradation.
Trimmer-2 kit is designed to normalize amplified full-length-enriched cDNA prepared using . The resulting cDNA contains equalized abundance of different transcripts and can be used for construction of cDNA libraries and for direct sequencing, including high-throughput sequencing on the next generation sequencing platforms (Roche/454, ABI/SOLiD or Illumina/Solexa). The kit also includes special adapters allowing use of Clontech SMART-based kits for construction of cDNA intended for Trimmer-2 normalization.
In-Fusion Cloning makes it easy to clone your SMARTer cDNA library into the pSMART2IF or pSMART2IFD linearized vectors (included in In-Fusion SMARTer kits) in just one 30 min reaction. Most importantly, since In-Fusion Cloning is designed to join fragments of DNA with 15 complementary bp at their ends, In-Fusion kits can be used to precisely transfer your SMARTer cDNA into ANY linearized vector. If you would like to clone your library into your own expression vector for functional analysis, simply amplify your vector by inverse PCR using primers that create linear vector ends that are complementary to the ends of the SMARTer cDNA. Primers must have two characteristics: the 5’ end of the primer must contain 15 bases that are complementary to 15 bases at one end of the DNA fragment to which the vector will be joined (i.e., the insert), and the 3’ end of the primer must contain sequence that is specific to the target vector.