Q: What is the principle of SMARTTM cDNA technology?
Answer: SMARTTM cDNA synthesis technology uses total RNA or Poly A RNA as a template, a modified oligo(dT) oligonucleotide as a primer, and is synthesized under the catalysis of MMLV reverse transcriptase. chain. When reverse transcriptase reaches the 5' end of the mRNA, the terminal transferase activity of the reverse transcriptase adds 3-5 deoxycytidylic acids to the 3' end of the first-strand cDNA. The 3' end of the SMART oligonucleotide contains an extended stretch of guanylic acid residues that anneal to enriched cytidylic acid residues to form an extended template. Reverse transcriptase then converts the template and continues elongation using the SMART oligonucleotide as a template. The single-stranded cDNA synthesized in this way has a sequence complementary to the SMART oligonucleotide at the 3' end and a sequence complementary to the oligo(dT) primer at the 5' end. These sequences serve as primer synthesis sites for the next step of long-distance PCR. As a result, double-stranded cDNA enriched with full-length sequences can be obtained.
Q: What are the differences between the SMART kit series products?
Answer: SMART cDNA Library Construction Kit is used to construct high-quality cDNA libraries from small amounts of RNA samples. The kit includes the reagents required for SMART cDNA synthesis and library construction into TriplEx2 or Creator donor vector pDNR-LIB. Because this kit is specifically designed for library construction, it cannot be used with the Clontech? PCR-Select? cDNA Subtractive Hybridization Kit.
The SMART PCR cDNA Synthesis Kit can be used with Clontech? PCR-Select? cDNA subtraction technology. Total RNA cannot be used directly for subtraction experiments, but it can be used to amplify high-quality cDNA through SMART PCR cDNA Synthesis Kit, which can be used for hybridization experiments. Similarly, small amounts of Poly A RNA can also be amplified using the SMART PCR cDNA Synthesis Kit. SMART cDNA can also be used to perform virtual Northern hybridization instead of standard Northern hybridization when you only have limited RNA samples. SMART PCR cDNA Synthesis Kit contains SMARTII oligonucleotides and cDNA synthesis primers. Both sequences have the RsaI site required for Clontech? PCR-Select subtraction. This kit can also be used to construct SMART cDNA libraries in other vectors of your choice.
SMART RACE cDNA Amplification Kit combines SMART cDNA synthesis technology and RACE methods. The advantage of this kit is that it can construct full-length cDNA without adapter ligation, has high sensitivity and is simple in method.
Q: Can SMART technology be used for rapid amplification of cDNA ends (RACE)?
Answer: Yes. With the further refinement of the new SMART RACE cDNA Amplification Kit, all the advantages of SMART technology can be used in RACE protocols.
Q: Why is there a certain limit on the number of PCR cycles during the SMART cDNA Synthesis process?
Answer: One of the unique features of SMART cDNA technology is the long-distance PCR amplification step. In order to maintain the gene representation of SMART cDNA, it is necessary to perform as few cycles as possible.
If the cycle number is too high and the amplification exceeds the exponential growth phase, ssDNA will accumulate, making it unsuitable for later cDNA operations such as cloning or subtraction. Furthermore, the higher the cycle number, the higher the chance of errors from the thermostable polymerase, and the fidelity of the PCR decreases accordingly. Therefore, in order to obtain high-quality cDNA, it is very important that the stable cycle number is within recognized parameters.
Q: What are the characteristics of SMARTTM library?
Answer: SMART cDNA library enriches full-length cDNA. The average insert size is 1.5-2 Kb. The largest insert we have ever cloned from a SMART library was a β-myosin cDNA larger than 6 kb. Even if total RNA is used as the initial material, ribosomal RNA will almost never be screened. Experimental results showed that analysis of 50 clones from the SMART cDNA library showed no ribosomal RNA clones. The gene fidelity of SMART cDNA libraries constructed with 50 ng of total RNA or 25 ng of poly A RNA was similar to that of libraries constructed with 5 ug of poly A RNA using the standard Gubler and Hoffman method.
Q: Why does the cDNA amplified by SMARTTM PCR have no rRNA sequence?
Answer: Although rRNA can be reverse transcribed during SMART first-strand cDNA synthesis, these sequences will not be amplified during the subsequent PCR process. This is because in most cases, the rRNA sequence is reverse transcribed through its own guidance, so the final cDNA fragment does not have 5' and 3' SMART specific sequences and cannot be amplified by exponential PCR.
Q: What is the minimum amount of RNA required that can be used as a template for SMART PCR cDNA amplification?
Answer: Although we can use SMART technology to construct libraries from 10ng of total RNA, we recommend that the starting material be at least 50ng of total RNA or 25ng of poly A RNA. This ensures that the SMART cDNA population is of higher quality and has stronger gene representation.
Q: Can I change the SMARTTM oligonucleotide sequence?
Answer: Any change in the oligonucleotide sequence will affect the efficiency of cDNA synthesis and amplification. The sequence and design of SMART II and SMART IV oligonucleotides are protected by patents.
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