SYNTHESIS OF cDNA EXPLAINED
cDNA, or complementary DNA, is a crucial molecule in molecular biology and biotechnology. It is synthesized from messenger RNA (mRNA) through the process of reverse transcription. The synthesis of cDNA involves several key steps, including RNA extraction, reverse transcription, and purification. Here is a comprehensive overview of the synthesis of cDNA:
1. RNA Extraction: The first step in cDNA synthesis is the extraction of RNA, particularly mRNA, from the biological sample of interest. This can be achieved using commercially available RNA extraction kits or traditional methods such as phenol-chloroform extraction. The extracted RNA serves as the template for cDNA synthesis.
2. Reverse Transcription: Reverse transcription is the process by which cDNA is synthesized from an RNA template. This process is catalyzed by the enzyme reverse transcriptase, which synthesizes a complementary DNA strand using the mRNA as a template. The enzyme also possesses RNase H activity, which degrades the RNA strand in the RNA-DNA hybrid, leaving behind a single-stranded cDNA molecule.
3. Second Strand Synthesis (Optional): In some cases, particularly when double-stranded cDNA is required, a second strand synthesis step may be included. This involves the use of DNA polymerase to synthesize a complementary DNA strand to the single-stranded cDNA obtained from reverse transcription.
4. Purification: Following cDNA synthesis, it is essential to purify the cDNA to remove any remaining RNA, enzymes, nucleotides, and other impurities. Purification methods may include ethanol precipitation, silica membrane-based purification kits, or column-based purification techniques.
5. Quality Assessment: Once purified, the synthesized cDNA should undergo quality assessment to ensure its integrity and suitability for downstream applications. Common methods for assessing cDNA quality include agarose gel electrophoresis, spectrophotometry to determine concentration and purity, and quantitative PCR (qPCR) to assess amplification efficiency.
6. Storage: Finally, the synthesized cDNA can be stored at -20°C or -80°C for long-term preservation. Proper storage conditions are crucial to maintain the stability and integrity of the cDNA for future use in various molecular biology experiments.
In summary, the synthesis of cDNA involves RNA extraction, reverse transcription, optional second strand synthesis, purification, quality assessment, and storage. This process yields a valuable tool for studying gene expression, cloning genes, creating recombinant DNA constructs, and various other applications in molecular biology and biotechnology.
