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BOC Sciences offers cDNA synthesis that uses RNA as a template to obtain cDNA by reverse transcription. The cDNA can then be used as a template for various downstream applications in RNA research.
cDNA (complementary DNA) is synthetic DNA whose base sequence is complementary to that of DNA. cDNA is synthesized by the action of RNA-dependent DNA polymerase (reverse transcriptase) in the presence of appropriate primers using RNA as a template, and after synthesizing a single-stranded cDNA and then treating it with a base to remove its corresponding RNA, it is then synthesized by the action of DNA-dependent DNA polymerase using the single-stranded cDNA as a template. After single-stranded cDNA is synthesized and its corresponding RNA is removed by alkali treatment, single-stranded cDNA is used as a template to synthesize double-stranded cDNA by the action of DNA-dependent or RNA-dependent DNA polymerase.
Fig.1 Procedure for cDNA synthesis. (Meis et al., 2009)
BOC Sciences provides cDNA synthesis services in which the synthesized first-strand cDNA can be widely used for second-strand cDNA synthesis for PCR amplification, hybridization, cDNA library creation, and full-length cDNA synthesis.
| Method | Specifications |
| Self-priming method | The 3' end of the synthesized single-stranded cDNA is able to form a short hairpin structure, which provides a ready-made primer for the synthesis of the second strand. The second strand of the cDNA is synthesized by using the E. coli DNA polymerase I Klenow fragment or reverse transcriptase after denaturation of the DNA-RNA hybridization strand from the product of the first-stranded synthesis reaction, and the loop is digested with S1 nuclease specific for the single strand, which can be further cloned. |
| Replacement synthesis method | In the presence of dNTP, RNAase H is utilized to create cuts and nicks in the mRNA strand of the hybridized strand. This results in a series of RNA primers for the synthesis of the second strand, which is synthesized under the action of E. coli DNA polymerase I. |
| Analysis method | Specifications |
| Gel Electrophoresis | The size and purity of the cDNA can be initially determined by the electrophoretic migration rate. |
| Real-time quantitative PCR (qPCR) | qPCR measures the expression level of a target gene and provides relative or absolute quantitative results. |
| Sequence Analysis | Sanger sequencing or high-throughput sequencing technology enables cDNA sequence analysis. |
Reference
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