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cDNA synthesis methods and precautions


cDNA synthesis includes cDNA first-strand and second-strand synthesis. All methods of synthesizing the first strand of cDNA are based on total RNA or isolated and purified mRNA as a template, using RNA-dependent reverse transcriptase catalyzed by Oligo (dT) or random primers. This article will introduce the method and precautions of cDNA synthesis.

Two methods of cDNA synthesis, are self-directed and displacement synthesis.

Self-guided method: The 3' end of the synthesized single-stranded cDNA can form a short hairpin structure, which provides ready-made primers for the synthesis of the second strand. When the DNA-RNA hybrid strand of the first-strand synthesis reaction product is denatured Use E. coli DNA polymerase I Klenow fragment or reverse transcriptase to synthesize the second strand of cDNA, and finally digest the loop with S1 nuclease specific for single-strand to further clone. However, this method is not easy to control and is prone to sequence deletion or rearrangement.

In the presence of dNTPs, RNase H is used to create nicks and gaps in the mRNA strand of the hybridized strand. Thereby, a series of RNA primers are produced, which become primers for synthesizing the second strand, and the second strand is synthesized under the action of E. coli DNA polymerase I. This method is very efficient, using the first-strand reaction product directly, without purification, and without the use of S1 nuclease to cleave single-stranded hairpin loops.

Precautions for cDNA Synthesis

(1) In order to maintain the integrity of RNA, an RNase inhibitor is usually added to the first-strand synthesis reaction system to inhibit RNase activity. (2) The selection of primers for cDNA synthesis is critical, and primers are selected according to different experimental needs. When a particular mRNA has difficulty copying its full-length sequence because it contains sequences that terminate reverse transcriptase, a non-specific primer, a random hexamer primer, can be used to copy the full-length mRNA. With this approach, all RNA molecules in the system act as first-strand cDNA templates, and PCR primers impart the desired specificity during amplification. Usually, 96% of the cDNA synthesized with this primer is derived from rRNA. Oligo (dT) primers are an mRNA-specific method. The most specific priming method is to use oligonucleotides containing the complementary sequences of the target RNA as primers. If two specific primers are used in the PCR reaction, the synthesis of the first strand can be initiated by the paired primer closest to the 3' end of the mRNA.Using such primers produces only the desired cDNA, resulting in a more specific PCR amplification.

Finally, cDNA synthesis has a wide range of applications, which can be used for cDNA library construction, analyzing gene transcription products, obtaining target genes, synthesizing cDNA probes, and constructing high-efficiency RNA transcription systems. It is necessary to master its experimental steps and methods.

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